LSD1 inhibition suppresses ASCL1 and de-represses YAP1 to drive potent activity against neuroendocrine prostate cancer.

Lysine-specific demethylase 1 (LSD1 or KDM1A ) has emerged as a critical mediator of tumor progression in metastatic castration-resistant prostate cancer (mCRPC). Among mCRPC subtypes, neuroendocrine prostate cancer (NEPC) is an exceptionally aggressive variant driven by lineage plasticity, an adaptive resistance mechanism to androgen receptor axis-targeted therapies. Our study shows that LSD1 expression is elevated in NEPC and associated with unfavorable clinical outcomes. Using genetic approaches, we validated the on-target effects of LSD1 inhibition across various models. We investigated the therapeutic potential of bomedemstat, an orally bioavailable, irreversible LSD1 inhibitor with low nanomolar potency. Our findings demonstrate potent antitumor activity against CRPC models, including tumor regressions in NEPC patient-derived xenografts. Mechanistically, our study uncovers that LSD1 inhibition suppresses the neuronal transcriptional program by downregulating ASCL1 through disrupting LSD1:INSM1 interactions and de-repressing YAP1 silencing. Our data support the clinical development of LSD1 inhibitors for treating CRPC - especially the aggressive NE phenotype. Statement of Significance: Neuroendocrine prostate cancer presents a clinical challenge due to the lack of effective treatments. Our research demonstrates that bomedemstat, a potent and selective LSD1 inhibitor, effectively combats neuroendocrine prostate cancer by downregulating the ASCL1- dependent NE transcriptional program and re-expressing YAP1.

Targeted delivery of cytotoxic proteins to prostate cancer via conjugation to small molecule urea-based PSMA inhibitors.

Prostate cancer cells are characterized by a remarkably low proliferative rate and the production of high levels of prostate-specific proteases. Protein-based toxins are attractive candidates for prostate cancer therapy because they kill cells via proliferation-independent mechanisms. However, the non-specific cytotoxicity of these potent cytotoxins must be redirected to avoid toxicity to normal tissues. Prostate-Specific Membrane Antigen (PSMA) is membrane-bound carboxypeptidase that is highly expressed by prostate cancer cells. Potent dipeptide PSMA inhibitors have been developed that can selectively deliver and concentrate imaging agents within prostate cancer cells based on continuous PSMA internalization and endosomal cycling. On this basis, we conjugated a PSMA inhibitor to the apoptosis-inducing human protease Granzyme B and the potent Pseudomonas exotoxin protein toxin fragment, PE35. We assessed selective PSMA binding and entrance into tumor cell to induce cell death. We demonstrated these agents selectively bound to PSMA and became internalized. PSMA-targeted PE35 toxin was selectively toxic to PSMA producing cells in vitro. Intratumoral and intravenous administration of this toxin produced marked tumor killing of PSMA-producing xenografts with minimal host toxicity. These studies demonstrate that urea-based PSMA inhibitors represent a simpler, less expensive alternative to antibodies as a means to deliver cytotoxic proteins to prostate cancer cells.

The influence of prednisone on the efficacy of docetaxel in men with metastatic castration-resistant prostate cancer.

BACKGROUND: Prednisone and other corticosteroids can provide palliation and tumor responses in patients with prostate cancer. The combination of docetaxel and prednisone was the first treatment shown to prolong survival in men with metastatic castration-resistant prostate cancer (mCRPC). Since the approval of docetaxel in 2004, additional treatments are available, including abiraterone, which is also administered with prednisone. Therefore, patients are increasingly likely to have prednisone therapy several times throughout their disease course, and the contribution of prednisone to the efficacy of docetaxel is unknown. METHODS: We conducted a retrospective study of patients with mCPRC treated with docetaxel at our institution between 2004 and 2014. Patients were divided into two cohorts based upon whether prednisone was co-administered with docetaxel. Cohorts were further stratified based upon prior prednisone (with abiraterone) or hydrocortisone (with ketoconazole) use. The primary end point was clinical/radiographic progression-free survival (PFS). The secondary end points were >50% PSA response rate and PSA progression-free survival (PSA PFS). A multivariable Cox regression model was constructed to determine whether prednisone use was independently predictive of PFS. RESULTS: We identified 200 consecutive patients for inclusion in the study: 131 men received docetaxel with prednisone and 69 received docetaxel alone. The docetaxel-prednisone cohort had superior PFS compared with the docetaxel-alone cohort (median PFS: 7.8 vs. 6.2 months, HR 0.68 (95% confidence interval (CI) 0.48-0.97), P=0.03). Prednisone use was associated with a reduced risk of progression on docetaxel in the propensity score-weighted multivariable Cox model (P=0.002). Among abiraterone- or ketoconazole-pretreated patients, no difference in PFS was observed between prednisone-containing and non-prednisone-containing docetaxel regimens (median PFS: 7.1 vs. 6.3 months, HR 0.96 (95% CI 0.59-1.57), P=0.87). CONCLUSIONS: The incorporation of prednisone potentially augments the efficacy of docetaxel in patients with mCRPC. We hypothesize that this advantage is limited to patients who have not previously received corticosteroids. Prospective confirmation is needed.

CCAAT/Enhancer binding protein ß controls androgen-deprivation-induced senescence in prostate cancer cells.

The processes associated with transition to castration-resistant prostate cancer (PC) growth are not well understood. Cellular senescence is a stable cell cycle arrest that occurs in response to sublethal stress. It is often overcome in malignant transformation to confer a survival advantage. CCAAT/Enhancer Binding Protein (C/EBP) ß function is frequently deregulated in human malignancies and interestingly, androgen-sensitive PC cells express primarily the liver-enriched inhibitory protein isoform. We found that C/EBPß expression is negatively regulated by androgen receptor (AR) activity and that treatment of androgen-sensitive cell lines with anti-androgens increases C/EBPß mRNA and protein levels. Accordingly, we also find that C/EBPß levels are significantly elevated in primary PC samples from castration-resistant compared with therapy-naive patients. Chromatin immunoprecipitation demonstrated enhanced binding of the AR to the proximal promoter of the CEBPB gene in the presence of dihydroxytestosterone. Upon androgen deprivation, induction of C/EBPß is facilitated by active transcription as evident by increased histone 3 acetylation at the C/EBPß promoter. Also, the androgen agonist R1881 suppresses the activity of a CEBPB promoter reporter. Loss of C/EBPß expression prevents growth arrest following androgen deprivation or anti-androgen challenge. Accordingly, suppression of C/EBPß under low androgen conditions results in reduced expression of senescence-associated secretory genes, significantly decreased number of cells displaying heterochromatin foci and increased numbers of Ki67-positive cells. Ectopic expression of C/EBPß caused pronounced morphological changes, reduced PC cell growth and increased the number of senescent LNCaP cells. Lastly, we found that senescence contributes to PC cell survival under androgen deprivation, and C/EBPß-deficient cells were significantly more susceptible to killing by cytotoxic chemotherapy following androgen deprivation. Our data demonstrate that upregulation of C/EBPß is critical for complete maintenance of androgen deprivation-induced senescence and that targeting C/EBPß expression may synergize with anti-androgen or chemotherapy in eradicating PC.

The effect of the frequency and duration of PSA measurement on PSA doubling time calculations in men with biochemically recurrent prostate cancer.

BACKGROUND: PSA doubling time (PSADT) is an attractive intermediate end point for assessing novel therapies in biochemically recurrent prostate cancer (BRPC). This study explores whether PSADT calculations are influenced by frequency/duration of PSA measurements, and whether statistical variability leads investigators to find false significant results. METHODS: In retrospective analyses of two BRPC cohorts: Johns Hopkins Hospital (JHH) patients who deferred therapy and placebo patients on a randomized clinical trial (RCT), we calculated changes in PSADT from early measurements to later measurements using subsets of available PSAs for patients with ≥6 and ≥9 PSAs. We simulated hypothetical single-arm trials using randomly selected, 50-patient subsets and simulated two-arm RCTs. RESULTS: JHH cohort (n=205) had median follow-up 58 months, median age 61 years and median Gleason 7. PSA variability changed with duration of PSA measurement as median within-patient PSADT increases for men with >6 PSAs ranged from 1.0 to 1.4 months by PSA subset while increases for men with ≥9 PSAs ranged from 3.9 to 4.1 months. Frequency of measurement did not change PSA variability as PSADT increase was unchanged when odd values were used instead of all values. Approximately 30% of JHH men experienced >200% increases in PSADT. Up to 62% of 50-patient single-arm simulations detected a significant PSADT change, whereas simulated RCTs did not. Results were supported in the RCT placebo cohort; 46% of patients experienced PSADT increases >200%. CONCLUSIONS: These data suggest that calculated PSADT in BRPC may naturally increase over time in the absence of therapy and may be influenced by duration of PSA follow-up. As a result, single-arm trials could show false significant increases despite the lack of active treatment of these patients. Placebo-controlled RCTs including clinical end points are recommended to screen novel agents in men with BRPC to mitigate bias because of natural PSADT variability.

A phase I/IIA study of AGS-PSCA for castration-resistant prostate cancer.

BACKGROUND: This first-in-human phase I/IIA study was designed to evaluate the safety and pharmacokinetics (PKs) of AGS-PSCA a fully human monoclonal antibody directed to prostate stem cell antigen (PSCA) in progressive castration-resistant prostate cancer. PATIENTS AND METHODS: Twenty-nine patients were administered infusions of AGS-PSCA (1-40 mg/kg) every 3 weeks for 12 weeks; 18 final patients received a 40-mg/kg loading dose followed by 20-mg/kg repeat doses. Primary end points were safety and PK. Immunogenicity, antitumor activity and circulating tumor cells were also evaluated. RESULTS: No drug-related serious adverse events were noted. Dose escalation stopped before reaching the maximum tolerated dose as target concentrations were achieved. Drug levels accumulated linearly with dose and the mean terminal half-life was 2-3 weeks across dose levels. The 40-mg/kg loading dose followed by repeated 20-mg/kg doses yielded serum drug concentrations above the projected minimum therapeutic threshold after two to three doses without excessive drug accumulation or toxicity. Significant antitumor effects were not seen. CONCLUSIONS: A 40-mg/kg loading dose followed by 20-mg/kg infusions every 3 weeks is the recommended phase II dose of AGS-PSCA. PSCA is a promising drug target and studies in prostate and other relevant solid tumors are planned.

Deletion of p53 in human mammary epithelial cells causes chromosomal instability and altered therapeutic response.

The TP53 tumor suppressor gene is the most commonly mutated gene in human cancers. To evaluate the biological and clinical relevance of p53 loss, human somatic cell gene targeting was used to delete the TP53 gene in the non-tumorigenic epithelial cell line, MCF-10A. In all four p53-/- clones generated, cells acquired the capability for epidermal growth factor-independent growth and were defective in appropriate downstream signaling and cell cycle checkpoints in response to DNA damage. Interestingly, p53 loss induced chromosomal instability leading to features of transformation and the selection of clones with varying phenotypes. For example, p53-deficient clones were heterogeneous in their capacity for anchorage-independent growth and invasion. In addition, and of clinical importance, the cohort of p53-null clones showed sensitivity to chemotherapeutic interventions that varied depending not only on the type of chemotherapeutic agent, but also on the treatment schedule. In conclusion, deletion of the TP53 gene from MCF-10A cells eliminated p53 functions, as well as produced p53-/- clones with varying phenotypes possibly stemming from the distinct chromosomal changes observed. Such a model system will be useful to further understand the cancer-specific phenotypic changes that accompany p53 loss, as well as help to provide future treatment strategies for human malignancies that harbor aberrant p53.

Combinatorial androgen receptor targeted therapy for prostate cancer.

Prostatic carcinogenesis is associated with changes in the androgen receptor (AR) axis converting it from a paracrine dependence upon stromal signaling to an autocrine-initiated signaling for proliferation and survival of prostatic cancer cells. This malignant conversion is due to gain of function changes in which the AR activates novel genomic (i.e. transcriptional) and non-genomic signaling pathways, which are not present in normal prostate epithelial cells. During further progression, additional molecular changes occur which allow these unique malignancy-dependent AR signaling pathways to be activated even in the low androgen ligand environment present following androgen ablation therapy. These signaling pathways are the result of partnering the AR with a series of other genomic (e.g. transcriptional co-activators) or non-genomic (e.g. steroid receptor co-activator (Src) kinase) signaling molecules. Thus, a combinatorial androgen receptor targeted therapy (termed CART therapy) inhibiting several points in the AR signaling cascade is needed to prevent the approximately 30,000 US males per year dying subsequent to failure of standard androgen ablation therapy. To develop such CART therapy, a series of agents targeted at specific points in the AR cascade should be used in combination with standard androgen ablative therapy to define the fewest number of agents needed to produce the maximal therapeutic anti-prostate cancer effect. As an initial approach for developing such CART therapy, a variety of new agents could be combined with luteinizing hormone-releasing hormone analogs. These include: (1) 5alpha-reductase inhibitors to inhibit the conversion of testosterone to the more potent androgen, dihydrotestosterone; (2) geldanamycin analogs to downregulate AR protein in prostate cancer cells, (3) 'bulky' steroid analogs, which can bind to AR and prevent its partnering with other co-activators/signaling molecules, and (4) small molecule kinase inhibitors to inhibit MEK, which is activated as part of the malignant AR signaling cascade.

A supramicromolar elevation of intracellular free calcium ([Ca(2+)](i)) is consistently required to induce the execution phase of apoptosis.

Many agents, such as the endoplasmic reticulum Ca(2+) ATPase inhibitor, thapsigargin, or the ionophore, ionomycin, induce apoptosis by transiently elevating [Ca(2+)](i). The role of [Ca(2+)](i) in apoptosis induced by agents that do not immediately increase [Ca(2+)](i), such as 5-FdUr, TGF beta-1, doxorubicin, or radiation, is far more controversial. In the present paper, [Ca(2+)](i) was measured continuously for 120 h. in prostate and bladder cancer cell lines exposed to these four agents: 5-FdUR, TGF beta-1, doxorubicin, or radiation. Each of them consistently induced a delayed [Ca(2+)](i) rise associated with the morphological changes that characterize the execution phase of apoptosis (i.e. rounding, blebbing). This [Ca(2+)](i) rise occurred in two consecutive steps (< or = 10 microM and >10 microM) and resulted from a Ca(2+) influx from the extracellular medium. This delayed supramicromolar [Ca(2+)](i) rise was also observed previously in breast, prostate and bladder cancer cell lines exposed to thapsigargin. This influx regulated transcriptional reprogramming of Gadd153 and is required to activate cytochrome c release, caspase-3 activation, loss of clonal survival and DNA fragmentation. When cells were maintained in low extracellular Ca(2+) media, these phenomena were temporarily delayed but occurred on return to normal Ca(2+) medium. Similarly, apoptosis could be delayed by overexpressing the Ca(2+)-binding proteins, Calbindin-D(28K) and parvalbumin. As this delayed >or = 10 microM [Ca(2+)](i) elevation was observed in a number of cell lines exposed to a variety of different agents, we conclude that such elevation constitutes a key and general event of apoptosis in these malignant cells.

Design, synthesis, and pharmacological evaluation of thapsigargin analogues for targeting apoptosis to prostatic cancer cells.

A series of thapsigargin (TG) analogues, containing an amino acid applicable for conjugation to a peptide specifically cleaved by prostate-specific antigen (PSA), has been prepared to develop the drug-moiety of prodrugs for treatment of prostatic cancer. The analogues were synthesized by converting TG into O-8-debutanoylthapsigargin (DBTG) and esterifying O-8 of DBTG with various amino acid linkers. The compounds were evaluated for their ability to elevate the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in TSU-Pr1 cells, their ability to inhibit the rabbit skeletal muscle SERCA pump, and their ability to induce apoptosis in TSU-Pr1 human prostatic cancer cells. The activity of analogues, in which DBTG were esterified with omega-amino acids [HOOC(CH(2))(n)()NH(2), n = 5-7, 10, 11], increased with the linker length. Analogues with 3-[4-(L-leucinoylamino)phenyl]propanoyl, 6-(L-leucinoylamino)hexanoyl, and 12-(L-serinoylamino)dodecanoyl were considerably less active than TG, and analogues with 12-(L-alaninoylamino)dodecanoyl and 12-(L-phenylalaninoylamino)dodecanoyl were almost as active as TG. The 12-(L-leucinoylamino)dodecanoyl gave an analogue equipotent with TG, making this compound promising as the drug-moiety of a PSA sensitive prodrug of TG.

Pan-trk inhibition decreases metastasis and enhances host survival in experimental models as a result of its selective induction of apoptosis of prostate cancer cells.

During the progression of prostate cancer, molecular changes occur resulting in the autocrine production of a series of neurotrophins by the malignant cells. This is coupled with expression of high-affinity cognate receptors for these ligands, termed trk receptors, by these cancer cells. The binding of the neurotrophins to their trk receptors activates the receptor's latent tyrosine kinase activity inducing a series of signal transduction pathways within these prostate cancer cells. These molecular changes result in the acquisition by prostate cancer cells of a restricted requirement for these trk signaling pathways for optimal survival. CEP-701 is an indolocarbazole compound specifically designed as a potent inhibitor (IC(50), 4 nM) of the tyrosine kinase activity of the trk receptors required for initiation of these survival pathways. In the present studies, the consequences of CEP-701 inhibition of these trk signaling survival pathways were tested in vivo using both rat (R3327 AT 6.3 and H) and human (TSU-pr1 and CWR-22Rv1) prostatic cancer models. These in vivo studies demonstrated that treatment with CEP-701 inhibits the growth of both rodent and human prostate cancers, without being toxic to the normal tissue including the host prostate. Because of this selective effect, CEP-701 inhibits metastasis and growth of both primary and metastatic sites of prostate cancer. Based upon this profile, long-term survival studies were performed using the slow-growing Dunning H rat prostate cancer model. For these latter studies, the dosing regimen was 10 mg CEP-701/kg/dose twice a day via gavage 5 days a week. This regimen maintains CEP-701 tumor tissue concentrations of 25-50 nM. Such chronic dosing increased (P < 0.001) the median survival of rats bearing the slow growing H prostate cancers from 408 days (395-432 days, 95% confidence interval) for the vehicle group (n = 18) to 566 days (497-598 days, 95% confidence interval) for the CEP-701-treated group (n = 24).

Activation of latent protease function of pro-hK2, but not pro-PSA, involves autoprocessing.

BACKGROUND: Human glandular kallikrein 2 (hK2) and prostate-specific antigen (PSA) are members of an extensive kallikrein family of proteases. Both proteases are secreted as zymogens or proenzymes containing a seven amino acid propeptide that must be proteolytically removed for enzymatic activation. The physiological proteases that activate pro-hK2 and pro-PSA are not known. METHODS: The pro-hK2 peptide sequence is Val-Pro-Leu-Ile-Gln-Ser-Arg (VPLIQSR). For PSA, the amino acid sequence of the propeptide is Ala-Pro-Leu-Ile-Leu-Ser-Arg (APLILSR). Fluorescent substrates were made by coupling these peptide sequences to 7-amino-4-methylcoumarin (AMC). The hydrolysis of the VPLIQSR-AMC and APLILSR-AMC substrates by hK2, PSA, and a panel of purified proteases was determined. RESULTS: HK2 readily cleaved the pro-hK2 peptide substrate VPLIQSR-AMC with a rate of hydrolysis that was approximately 8-fold higher than an equimolar amount of purified trypsin. HK2 also had the highest hydrolysis rate from among a group of other trypsin-like proteases. In contrast, neither hK2 nor PSA was able to appreciably cleave the pro-PSA substrate APLILSR-AMC. The pro-PSA substrate was most readily hydrolyzed by urokinase and trypsin. CONCLUSIONS: HK2 can hydrolyze the pro-hK2 substrate suggesting that maturation of pro-hK2 to enzymatically active hK2 involves autoprocessing. As expected, PSA, a chymotrypsin-like protease, was unable to hydrolyze either of the propeptide substrates. Therefore, it is unlikely that PSA can auto-process its own enzymatic function. HK2 has trypsin-like specificity but was unable to hydrolyze the pro-PSA substrate. These results raise the possibility that an additional processing protease may be required to fully process PSA to an enzymatically active form.

Concentration of enzymatically active prostate-specific antigen (PSA) in the extracellular fluid of primary human prostate cancers and human prostate cancer xenograft models.

BACKGROUND: Prostate-specific antigen (PSA) targeted prodrugs are under development in our laboratory. Concentrations of total PSA and enzymatically active PSA produced by various human prostate cancer xenograft models have not been well characterized. METHODS: The concentration of PSA secreted into the extracellular fluid (ECF) in normal human prostate tissue, primary prostate cancers obtained directly from patients, and serially passageable human prostate cancer xenografts (PC-82, LNCaP, LAPC-4) were determined using Tandem assays. Percent enzymatically active PSA in the ECF and in conditioned media was also determined using a previously validated assay employing a monoclonal antibody to the PSA catalytic site. In addition, the concentration and activity of PSA within sera from men with and without prostate cancer, as well as from tumor-bearing animals, was likewise assayed. RESULTS: Normal human prostate tissue and primary human prostate cancers have high concentrations of PSA in the ECF (i.e., 1600-2100 nM). The majority of this PSA is enzymatically active (i.e., 80-90%). Human PC-82 prostate cancer xenografts also have high concentrations of PSA in the ECF (624 +/- 360 nM), and the majority of this PSA is also enzymatically active (i.e., 66 +/- 4%). In contrast, much lower concentrations of PSA are found in the ECF from LNCaP (45 +/- 9 nM) and LAPC-4 (7.3 +/- 0.6 nM). Only a small portion of the total PSA isolated from DHT-containing, serum-free, conditioned media from these cell lines is enzymatically active (i.e., approximately 18%). While PSA was detected in all serum samples regardless of the type of host, no enzymatically active PSA was detected in any of these serum samples. CONCLUSIONS: Prostate cancers obtained directly from patients produce and secrete large amounts of PSA, the majority of which is highly enzymatically active. In contrast, while PSA was detected in the sera, none of this PSA was enzymatically active. This is also the case for the human PC-82 prostate cancer xenografts. In contrast, LNCaP and LAPC-4 human prostate cancer xenograft models secrete approximately 70-300-fold less PSA in the ECF than prostate cancers from patients and the majority of this PSA is enzymatically inactive. Also, the serum from these animals had detectable PSA, but none of this PSA was enzymatically active. Thus, these latter two prostate cancer models define the least and the PC-82, the most, optimized xenograft model for screening PSA targeted prodrugs.

Expression of prostate-specific membrane antigen in transitional cell carcinoma of the bladder: prognostic value?

The expression of Prostate-specific membrane antigen (PSMA) mRNA was assessed in the normal bladder urothelium (n = 9), transitional cell carcinoma (TCC) specimens (n = 52), TCC-derived cell lines (n = 3), and preoperative blood samples from TCC patients (n = 27). Specific PSMA mRNA was found in 100% of normal and malignant tissues and two cell lines. PSMA protein was detected in normal (n = 3) and malignant tissues (n = 4). Using a PSMA-specific substrate, PSMA enzymatic activity was found in two bladder cell lines and correlated with immunostaining. Seven of the 27 TCC preoperative blood samples were positive by reverse transcription-PCR. These preliminary results, obtained on a nonrandomized cohort of patients, correlated with tumor invasion (positive RT-PCR: 0% for pT < or = 2 versus 41% for pT > or = 3) and 2-year survival rate (81% in the PSMA-negative group versus 29% in the PSMA-positive group). Although the clinical usefulness of this assay requires confirmation in larger prospective randomized trials, current preliminary results suggest that a blood-borne PSMA mRNA PCR assay may be a useful tool to predict a poor outcome in TCC patients.

In vivo activity of a PSA-activated doxorubicin prodrug against PSA-producing human prostate cancer xenografts.

BACKGROUND: There is currently no effective therapy for men with metastatic prostate cancer who relapse after androgen ablation. Prolonged administration of effective concentrations of standard chemotherapeutic agents is usually not possible because of dose-limiting systemic toxicities. A new strategy to target cytotoxic agents specifically to sites of metastatic prostate cancer while avoiding systemic toxicity would be to develop prodrugs that are inactive when given systemically but become activated when processed proteolytically within prostate cancer metastases by prostate-specific antigen (PSA). In this study, the in vivo activity of a prodrug consisting of doxorubicin (Dox) conjugated to a PSA-specific peptide carrier is described. METHODS: Nude mice bearing PSA-producing human prostate cancer xenografts were treated either intraperitoneally (IP) or by continuous infusion with the Dox prodrug. Toxicity (weight loss, death) and antitumor efficacy (tumor volume changes) were determined. RESULTS: The PSA-peptide Dox prodrug had no discernible systemic toxicity when given at four times the 100% lethal Dox equivalent dose. An IP dose of 60 mg/kg/week x 4 weeks resulted in a 57% decrease in tumor weight vs. control after 40 days. A 25 mg/kg/week dose given by continuous infusion produced a similar decrease in tumor weight vs. control. CONCLUSIONS: The PSA-specific peptide/doxorubicin prodrug can be used to deliver higher intratumoral levels of Dox for longer duration while avoiding systemic toxicity. In addition, these results validate the specificity of the PSA-specific peptide as a targetable drug carrier. This PSA-specific peptide could also be used as a carrier to target a wide variety of cytotoxic agents for specific activation within sites of metastatic prostate cancer.

Delayed micromolar elevation in intracellular calcium precedes induction of apoptosis in thapsigargin-treated breast cancer cells.

Thapsigargin (TG), a highly specific inhibitor of the sarcoplasmic reticulum and endoplasmic reticulum Ca2+-ATPase pump, can induce apoptosis in a variety of epithelial and lymphoid cell types. In prostate cancer cell lines, TG induces an initial 5- to 10-fold elevation of intracellular calcium ([Ca2+]i) within a few minutes of exposure. With prolonged exposure times (i.e., 12-36 h) a second elevation of [Ca2+]i to >10 microM is observed. In this study, the human breast carcinoma cell lines MCF-7 and MDA MB 468 cells were used to determine the temporal relationship between TG-induced elevation of [Ca2+]i and activation of programmed cell death. Using a microinjection method that allows for long-term analysis of [Ca2+]i changes, we found that after TG exposure, calcium measurements in these cells demonstrated an initial rise (>4-fold) in [Ca2+]i that occurred within minutes and returned to baseline within a few hours. With prolonged TG exposure, the cells underwent a second elevation (>5 microM) of [Ca2+]i occurring stochastically between 12 and 36 h after the initial exposure to TG. Both of the cell lines were growth-inhibited by 100 nM TG after only 1 h of exposure, but clonogenic ability in the MCF-7 cells was significantly reduced only after 48 h of exposure. The induction of apoptosis by TG was demonstrated by morphological changes typical for programmed cell death and DNA fragmentation (both high molecular weight and oligonucleosomal-sized fragments were detected) after 48 h of treatment. TG induction of apoptosis in these breast cancer cells occurred subsequent to the secondary rise in [Ca2+]i, which confirmed that this secondary rise in [Ca2+]i is not prostate cancer-specific. The secondary rise in [Ca2+]i to micromolar levels may directly activate the endonucleases responsible for DNA fragmentation that occurs as part of the apoptotic process. These studies indicate that TG is an active agent in vitro against breast cancer cells. Inactive prodrug analogues of TG are currently being developed that can be activated by tissue-specific proteases, and further pursuit of this strategy as a potential treatment for breast cancer is warranted.

Complete androgen blockade for prostate cancer: what went wrong?

PURPOSE: We summarized and critically assessed all available data from phase III clinical trials on complete androgen blockade versus surgical or medical castration alone. MATERIALS AND METHODS: Published results in journals and abstracts of phase III trials, and published meta-analyses were reviewed. We also reviewed quality of life and toxicity issues associated with the addition of antiandrogens to medical or surgical castration. Finally, we discuss the original rationale for complete androgen blockade in the context of current knowledge. RESULTS: A total of 27 clinical trials using various combinations of androgen deprivation were identified, of which 3 showed a statistically significant benefit for the complete androgen blockade arm. There were 5 publications of meta-analyses that each used different selection criteria for the inclusion of studies in the final analysis. Toxicity and quality of life have not been widely investigated in prospective fashion but the available data suggest a higher toxicity rate and decreased quality of life with complete androgen blockade. CONCLUSIONS: The extensive body of data does not support routine use of antiandrogens in combination with medical or surgical castration as first line hormonal therapy in patients with metastatic prostate cancer.

Thapsigargin induces a calmodulin/calcineurin-dependent apoptotic cascade responsible for the death of prostatic cancer cells.

BACKGROUND: New agents are required for the treatment of androgen-independent prostate cancer. Due to the low rate of proliferation of these malignant cells, agents which can activate the apoptotic death of these cells without requiring the cells being in the proliferative cell cycle are critically required. Thapsigargin (TG), via its ability to perturb intracellular free calcium [Ca(2+)](i), is such a cell proliferation-independent cytotoxic agent. The present study focuses on more completely describing the biochemical cascade during the apoptotic death of androgen-independent prostate cancer cells induced by TG and on the mechanistic requirements for this death. METHODS: A variety of cell and molecular biology techniques (e.g., time-lapse video, fluorescence image analysis, Northern and Western blotting) were used to examine the temporal relationship between changes in [Ca(2+)](i), GADD 153 transcription, translocation of the NFATc transcription factor to the nucleus, translocation of BAD from the cytosol to the mitochondria, caspase 9 activation, DNA fragmentation, and the loss of clonogenic survival induced by TG treatment of both human TSU-prl and rat AT3.1 prostate cancer cells in vitro. Additional studies using both microinjection of inhibitors of calmodulin and DNA transfections to induce expression of Ca(2+) binding proteins, e.g., calbindin, were performed to evaluate the causal relationship between [Ca(2+)](i) elevation, calmodulin/calcineurin activation, and apoptosis of prostate cancer cells. RESULTS: Using simultaneous fluorescence ratiometric and phase contrast image analysis in individual cells followed longitudinally for several days, it was documented that TG induced early (1-12 hr) moderate (i.e., <500 nM) elevation in [Ca(2+)](i). During this early rise in [Ca(2+)](i), genes like GADD 153 are induced at the transcriptional level. This early rise is followed by a return of [Ca(2+)](i) to baseline (i.e., approximately 50 nM) before the induction of a delayed (i.e., >12 hr) secondary rise ( approximately 10 microM) in [Ca(2+)](i). During the secondary rise in [Ca(2+)](i), Ca(2+) binds to calcineurin and calmodulin, allowing these proteins to form a complex which activates calcineurin's latent phosphatase activity. Once activated, calcineurin dephosphorylates NFATc and BAD, allowing translocation of these proteins to the nucleus and mitochondria, respectively. BAD translocation induces the release of cytochrome C from the mitochondria into the cytoplasm, which results in activation of caspase 9 and DNA fragmentation. If the TG-induced rise in [Ca(2+)](i) is blocked by overexpressing calbindin, or if calmodulin function is inhibited, these apoptotic events are prevented. CONCLUSIONS: TG induces the apoptotic death of prostate cancer cells via the activation of a reversible signaling phase induced by a transient nanomolar rise in [Ca(2+)](i), which involves new gene transcription and translation. This reversible signaling phase is followed by an irreversible commitment to undergo the execution phase which is induced by a secondary micromolar rise in [Ca(2+)](i). This secondary [Ca(2+)](i) rise irreversibly commits the cell to a calmodulin/calcineurin-dependent cascade, which results in DNA and cellular fragmentation into apoptotic bodies.

Inhibition of caspase activity does not prevent the signaling phase of apoptosis in prostate cancer cells.

BACKGROUND: Caspases are a family of cysteine proteases capable of characteristically cleaving after an aspartic acid residue. Various members of the caspase family (e.g., caspases 8 and 9) have been implicated as critical initiators in the signaling phase, while others (e.g., caspases 3, 6, and 7) have been implicated in the effector or execution phase of apoptosis. Thapsigargin (TG) is capable of inducing cell proliferation-independent apoptosis of prostate cancer cells. This study was undertaken to determine if caspase inhibition can prevent TG- or 5-fluorodeoxyuridine (5-FrdU)-induced apoptosis in prostate cancer cells. METHODS: Caspase activity was evaluated by Western blot analysis of the cleavage of retinoblastoma (Rb) protein, a caspase substrate during TG-induced death of prostate cancer cells. In addition, hydrolysis of caspase-specific fluorescent peptide substrates was assayed in lysates from TG-treated cells. Clonogenic survival assays were performed following treatment of rat AT3 and human TSU-Pr1 prostate cancer cell lines with TG and 5-FrdU in the presence and absence of peptide caspase inhibitors. AT3.1 cells transfected with the crmA gene, encoding a viral protein with caspase-inhibitory activity, were also tested for clonogenic survival following TG and 5-FrdU exposure. RESULTS: During treatment with TG, Rb is first dephosphorylated and then proteolytically cleaved into 100-kDa and 40-kDa forms, indicative of caspase activity. A 6-8-fold increase in class II (i.e., caspases 3, 7, and 10) hydrolysis of the caspase substrate Z-DEVD-AFC was observed after 24 hr of TG or 5-FrdU. AT3 cells expressing crmA (i.e., an inhibitor of caspases 1, 4, and 8) were not protected from apoptosis induced by TG or 5-FrdU. The caspase inhibitors Z-DEVD-fmk (i.e., an inhibitor of caspases 3, 7, and 10) and Z-VAD-fmk (i.e., a general caspase inhibitor) were also unable to protect TSU and AT3 cells from apoptosis induced by TG or 5-FrdU. CONCLUSIONS: Caspase activation may play a role in the downstream effector phase of the apoptotic cascade; however, in this study, caspase inhibition did not prevent the signaling phase of apoptosis induced by two agents with distinct mechanisms of cytotoxicity, TG or 5-FrdU. These results suggest that caspase inhibition by recently described endogenous caspase inhibitors should not lead to development of resistance to TG. A strategy for targeting TG's unique cytotoxicity to metastatic prostate cancer cells is currently under development.