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.

KAI1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2.

A gene from human chromosome 11p11.2 was isolated and was shown to suppress metastasis when introduced into rat AT6.1 prostate cancer cells. Expression of this gene, designated KAI1, was reduced in human cell lines derived from metastatic prostate tumors. KAI1 specifies a protein of 267 amino acids, with four hydrophobic and presumably transmembrane domains and one large extracellular hydrophilic domain with three potential N-glycosylation sites. KAI1 is evolutionarily conserved, is expressed in many human tissues, and encodes a member of a structurally distinct family of leukocyte surface glycoproteins. Decreased expression of this gene may be involved in the malignant progression of prostate and other cancers.

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.

Macrophage role in the anti-prostate cancer response to one class of antiangiogenic agents.

BACKGROUND: Tumor-associated macrophages (TAMs) can either promote angiogenesis (i.e., the formation of new blood vessels) in tumors by secreting tumor necrosis factor-alpha (TNF-alpha) or inhibit angiogenesis by producing granulocyte-macrophage colony-stimulating factor (GM-CSF), which in turn stimulates production of the antiangiogenic protein plasminogen activator inhibitor type 2 (PAI-2). We tested, alone or in combination, the anti-prostate cancer activity of agents that perturb macrophage function. METHODS: By use of enzyme-linked immunosorbent assays, we measured the effects of Linomide (roquinimex), thalidomide, pentoxifylline, and genistein on TNF-alpha and GM-CSF production in vitro by virally transformed RAW 264.7 mouse macrophages and on PAI-2 production in vitro by human macrophages. The antitumor effects of these agents were tested in vivo on transplanted Dunning R-3327 MAT-Lu rat prostate cancers; TAM numbers and blood vessel densities in these cancers were determined by use of immunocytochemistry. RESULTS: Linomide selectively inhibited mouse macrophage secretion of TNF-alpha but not of GM-CSF; however, thalidomide, pentoxifylline, and genistein inhibited the production of both cytokines. Linomide, but not thalidomide or pentoxifylline, increased production of PAI-2 by human macrophages. When administered to rats bearing MAT-Lu tumors, each of the tested agents reduced TAM numbers (Linomide, by 46%; thalidomide, by 94%; pentoxifylline, by 71%; and genistein, by 96%). However, all of the agents reduced tumor blood vessel density and tumor growth, with Linomide being the most effective (44% reduction in blood vessel density and 69% inhibition of tumor growth). None of the other agents potentiated Linomide's antitumor effect. CONCLUSIONS: Linomide is unique among the antiangiogenic agents tested, in that it inhibits the stimulatory effects of TAMs on tumor angiogenesis without eliminating their antiangiogenic effects, and may thus prove to be more effective against prostate cancer.

Activation of programmed cell death by recombinant human tumor necrosis factor plus topoisomerase II-targeted drugs in L929 tumor cells.

The mechanism of death induced by recombinant human tumor necrosis factor (rHuTNF) in L929 tumor cells of C3H mice was investigated. Treatment with rHuTNF led to fragmentation of DNA into nucleosomal oligomers and to induction of the expression of TRPM-2, a programmed cell death-associated gene. Both events preceded cell death by several hours. Treatment with DNA topoisomerase II inhibitors accelerated both the rHuTNF-mediated DNA fragmentation and the elevation in TRPM-2 messenger RNA levels. These results suggest that rHuTNF exerts its cytotoxicity on L929 cells by activating programmed cell death, leading to apoptosis, and that topoisomerase II inhibitors enhance rHuTNF-mediated cytotoxicity by accelerating this process.

Inheritance of a genetic factor from the Copenhagen rat and the suppression of chemically induced mammary adenocarcinogenesis.

Female rats of ten different inbred strains were tested for their susceptibility to 7, 12-dimethylbenz[a]anthracene (DMBA)-induced mammary adenocarcinogenesis. Five of these strains (OM, NSD, WF, LEWIS, BUFF) were demonstrated to be highly susceptible to DMBA exposure developing greater than 2 continuously growing, macroscopically detectable mammary adenocarcinomas/rat following gastric intubation with DMBA. In contrast, the COP female rat is completely resistant. Cross-breeding COP to any of the highly susceptible strains produced F1 hybrids which are resistant to DMBA-induced mammary adenocarcinogenesis. Cross-breeding the same highly susceptible strains to the Fischer strain (i.e., a strain only intermediately susceptible to DMBA) produced F1 hybrids which were just as highly inducible as their highly susceptible non-Fischer parental strain. The resistance of the F1 hybrids produced by cross-breeding these highly susceptible strains to the COP strain therefore is not due to a recessive lack of susceptibility but to an active process of suppression of the high susceptibility of these F1 hybrids to DMBA-induced mammary adenocarcinogenesis. To determine if the site of action of the COP gene responsible for this suppression is in the mammary gland itself or at the host systemic level, a series of factors (i.e., host growth rate, mammary gland growth rate, systemic hormone level during the estrus cycle, serum and tissue levels of DMBA and its metabolites) were compared between female rats of the highly susceptible NSD versus the resistant COP strain. These results suggested that host systemic factors are not involved. To test this directly, donor mammary glands from the highly susceptible NSD and the resistant COP strains were transplanted into F1 hybrids produced by cross-breeding these two strains. The resultant donor glands were then directly exposed to DMBA, the animals were followed, and the incidence of mammary adenocarcinomas was determined. Seventy % of the NSD donor glands developed continuously growing cancers while only 10% of COP donor glands did the same. These results are clearly incompatible with host systemic factors being the major determinant in the resistance of NSD X COP F1 hybrids to DMBA-induced mammary adenocarcinogenesis. Instead, these results directly demonstrate that it is the genetic makeup of the donor mammary gland itself which determines its response to DMBA exposure.

Relationship between tumor size and curability of prostatic cancer by combined chemo-hormonal therapy in rats.

Nearly all men with metastatic prostatic cancer respond to androgen ablation, demonstrating that at least a portion of their cancer cells are androgen responsive. Unfortunately, however, individual prostatic cancers contain clones of androgen-independent, in addition to androgen-responsive, cancer cells. Due to this tumor cell heterogeneity, essentially all patients treated with androgen ablation alone eventually relapse to a state unresponsive to further antiandrogen therapy; cures are rarely produced. To produce cures, additional nonhormonal therapy targeted at the androgen-independent prostatic cancer cells within the patient should be combined with androgen ablation targeted at the androgen-dependent cancer cells. The validity of such combined chemo-hormonal therapy was tested using, as the experimental model, two members of the Dunning system of serially transplantable rat prostatic cancers. Specifically the slow growing, well differentiated H and the fast growing, poorly differentiated G Dunning sublines were used, since these cover the clinical extremes observed for human prostatic cancers. The chemotherapeutic agent used in combination with surgical androgen ablation (i.e., castration) in these studies was Cytoxan. These studies demonstrate that for both the H and G cancer-bearing rats, the mean survival following combined chemo-hormonal therapy was increased above that found for castrate or Cytoxan when either was used as monotherapy. In addition, an inverse relationship between tumor size at the time of initiation of therapy and the ability of the combined chemo-hormonal therapy to cure animal bearing either the H or G sublines was demonstrated. Such combined chemo-hormonal therapy could only cure a proportion (i.e., 30-40%) of H or G tumor-bearing animals if initiated when the tumor was less than or equal to 0.2 cm3 in size. In contrast, if the tumor was 1-2 cm3 in starting size when the chemo-hormonal therapy was initiated, no animal was cured. Neither of the monotherapies (i.e., castrates or Cytoxan alone) could cure any animals regardless of the starting size.

The aging ACI/Seg versus Copenhagen male rat as a model system for the study of prostatic carcinogenesis.

In untreated male ACI/Seg rats, the incidence of microscopic cancer of the ventral prostate is zero below the age of 16 months but increases to greater than 80% by 36 months of age, while the incidence of grossly manifest cancer of the ventral prostate is zero before the age of 20 months and increases to 16% by 36 months of age. In contrast, in untreated male Copenhagen rats, the incidence of microscopic prostatic cancer is only 10%, and the incidence of grossly manifest prostatic cancer is less than 1% even at 36 months of age. Analysis of the relationship between ACI/Seg host age and the incidence of microscopic versus grossly manifest prostatic cancer suggests that microscopic prostatic cancer is the result of a multistep process and that additional events are required for the progression of microscopic to grossly manifest prostatic cancer. Comparison of the serum levels of sex steroid hormones between aging male ACI/Seg and Copenhagen rats reveals substantial differences, suggesting that alterations in the serum testosterone:estrogen ratio may be one of the factors involved in prostatic carcinogenesis in the ACI/Seg rat. Chronic elevation of serum testosterone levels in these male ACI/Seg rats by means of exogenous testosterone treatment alone, however, does not induce the precocious development of microscopic prostatic cancer in young (i.e., less than 1 year of age) animals, suggesting that other factors, in addition to alterations in serum levels of testosterone, are required for prostatic carcinogenesis in the male ACI/Seg rat.

A mammary cancer suppressor gene and its site of action in the rat.

Fifty-day-old female rats of the inbred Osborne-Mendel (OM) and Copenhagen (COP) strains were exposed to a single dose of either of 2 highly effective mammary chemical carcinogens, 7,12-dimethylbenz[a] anthracene (DMBA) or 1-methyl-1-nitrosourea (MNU). Female OM rats are highly susceptible to both of these carcinogens developing greater than 5 mammary adenocarcinomas per rat following a single exposure to either chemical. In contrast, female COP rats are completely resistant to both DMBA and MNU mammary cancer induction. Genetic breeding analysis of the F1 and F2 hybrids produced by crossing COP to OM rats demonstrated that the resistance of the female COP rat to DMBA and MNU is due to the presence of a single dominant autosomal allele in the germ line of the COP rat. Transplantation experiments demonstrated that the site of action of this COP gene is within the mammary epithelial cells themselves, not systemically or at the local mammary gland level within nonepithelial mammary cells. The resistance to DMBA-induced mammary carcinogenesis affected by the COP gene does not involve prevention of the initial interaction of DMBA with the mammary epithelial cells, but suppression of the progression of these initiated mammary cells to full cancer. This suppression does not involve paracrine release of diffusible factor(s). This gene does not suppress the development of MNU-induced renal or bladder cancers in the COP female rats. Thus, this newly identified autosomal dominant gene is specifically a mammary cancer suppressor gene. Analysis of the response of female feral rats to DMBA or MNU exposure demonstrates that this mammary cancer suppressor gene is also functional in feral rats. This suggests that this mammary cancer gene is functionally inactivated either by mutation or deletion in the germ line of highly susceptible strains of rats like the OM and inbred Sprague-Dawley rats, but functionally retained in resistant strains like the COP.

Genetic control of resistance to chemically induced mammary adenocarcinogenesis in the rat.

Fifty-day-old female rats of a series of outbred (i.e., SD) and inbred (i.e., NSD, WF, LEW, F344, ACI, and COP) strains were exposed to a single dose of either of two highly effective mammary chemical carcinogens, 7,12-dimethylbenz[a]anthracene (DMBA) or 1-methyl-1-nitrosourea (MNU), to determine the characteristic number of mammary adenocarcinomas induced/rat for each strain. Female rats of the inbred NSD, WF, and LEW strains were found to be as highly susceptible to DMBA exposure as the randomly outbred SD strain (i.e., greater than 2 mammary adenocarcinomas/rat develop). Inbred female F344 and ACI rats were found to be much less susceptible to DMBA induced mammary adenocarcinogenesis (i.e., less than 1.2 mammary adenocarcinomas/rat). In contrast to all the other inbred strains, the female COP rat was unique in that it is essentially completely resistant to all attempts to induce mammary adenocarcinomas by either DMBA or MNU exposure. Genetic breeding analysis demonstrated that the resistance of the mammary epithelium of the female COP rat to DMBA and MNU is due to the mendelian inheritance of a dominant, autosomal genetic allele. The inheritance of a single copy of this resistance allele is able to prevent both the DMBA and MNU induced development of mammary adenocarcinomas in F1 hybrids produced by cross-breeding COP to the highly susceptible NSD animal.

Determination of the number of events required for mammary carcinogenesis in the Sprague-Dawley female rat.

Female Sprague-Dawley rats were exposed to a single, graded dose of either of two highly effective mammary chemical carcinogens, 7,12-dimethylbenzanthracene (DMBA) or N-methylnitrosourea, in order to determine the number of mammary cancers per rat induced by a range of carcinogenic doses. These data were then used to separately construct dose-response curves characteristic for DMBA- and N-methylnitrosourea-induced mammary carcinogenesis. Analysis of these characteristic dose-response curves demonstrated that, following a single exposure to either DMBA or N-methylnitrosourea, the number of mammary cancers per rat increased not linearly but as the second power of dose of carcinogen used. These results are clearly incompatible with mammary carcinogenesis being a single step process in the female Sprague-Dawley rat. In direct contrast these results are entirely consistent with a malignant process requiring two transformation events. When female Sprague-Dawley animals are exposed multiple times to a suboptimal dose of DMBA, the number of mammary cancers induced per rat increases synergistically, not merely additively, as compared to a single dose exposure. Again this result is consistent only with mammary carcinogenesis requiring at least two transformation events.

Hormonally responsive versus unresponsive progression of prostatic cancer to antiandrogen therapy as studied with the Dunning R-3327-AT and -G rat adenocarcinomas.

The present study has compared the response to antiandrogen therapy of the serially transplantable Dunning R-3327-AT (hereafter called AT) versus Dunning R-3327-G (hereafter called G) rat prostatic adenocarcinoma. Castration or chemical antiandrogen therapy (i.e., cyproterone acetate and diethylstilbestrol) of rats bearing established AT or G tumors results in neither regression of tumor volume nor a cessation of the continuous growth of either tumor. By these criteria, both the AT and G tumors progress following antiandrogen therapy. For the AT tumor, this progression is completely unresponsive to hormonal therapy, and thus such therapy does not increase survival of AT tumor-bearing rats. The AT tumor is therefore an example of hormonally unresponsive progression. In direct contrast, while the G tumor likewise progresses following antiandrogen therapy, this therapy does induce a 1.8-fold decrease in the subsequent growth rate of the G tumor. This positive response during progression of the G tumor results in a 78% increase in the survival of G tumor-bearing rats treated with antiandrogen therapy. The G tumor is therefore an example of hormonally responsive progression. These results indicate neither that prostatic cancers which do not regress or cease growing following antiandrogen therapy can necessarily be considered hormonally unresponsive nor that antiandrogen therapy of such tumors has been completely ineffective, since, as shown in the present study, such progression can be of either a hormonally unresponsive or a responsive type. Regardless of which type of progression occurs, however, additional therapy is required to further increase survival. The present study demonstrates that such additional therapy should probably not include the subsequent use of pharmacological doses of exogenous androgen, since, depending on the type of progression, such treatments can actually decrease survival.

Biochemical methods for predicting metastatic ability of prostatic cancer utilizing the dunning R-3327 rat prostatic adenocarcinoma system as a model.

At present, there is no established diagnostic method by which the metastatic ability of an individual prostatic cancer can be accurately predicted. Metastasis is a multistep process, the first critical step of which is invasion. Tumor invasion has been suggested to involve a variety of hydrolytic enzyme activities; therefore, the tumor levels of these activities might be indicative of the overall metastatic ability of the cancer. In order to evaluate if the quantitative levels of hydrolytic enzymes can be used to predict the metastatic ability of individual prostatic cancers, five different Dunning R-3327 rat prostatic adenocarcinoma sublines, with widely varying metastatic abilities, were assayed for the respective levels of a variety of hydrolytic enzyme activities (collagenase, trypsin-like, cathepsin B, neutral protease, N-acetyl-beta-glucosaminidase, chymotrypsin-like, leucine aminopeptidase, elastase, and plasminogen activator). These studies demonstrated that most hydrolytic activities are not elevated when going from normal prostate to prostatic cancer. In addition, only the levels of elastase and chymotrypsin-like activity were found to be consistently higher in highly metastatic prostatic cancers than in either the normal prostate or low-metastatic prostatic cancers. It was found that, by combining the relative activities of elastase and chymotrypsin-like activity and then dividing by the relative activities of N-acetyl-beta-glucosaminidase, a biochemical metastatic index could be constructed which accurately reflected the respective metastatic ability of the Dunning sublines.

Potentiation of the antiangiogenic ability of linomide by androgen ablation involves down-regulation of vascular endothelial growth factor in human androgen-responsive prostatic cancers.

Linomide is a p.o. active antiangiogenic agent that has been demonstrated to be effective in suppressing the in vivo growth of rat and human prostatic cancer xenografts. The present studies were conducted to determine whether the angiogenic molecules, vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) and basic fibroblast growth factor (bFGF) are expressed in vitro by DU-145, PC-3, TSU-PR1, and LnCaP human prostate cancer cell lines and whether Linomide inhibits the secretion of these angiogenic molecules. Additionally, two different androgen-responsive human prostatic cancer xenograft models (i.e., PC-82 and A-2) were used to determine whether androgen ablation-induced reduction in tumor growth is associated with a reduction in tumor VEGF and/or bFGF levels. These studies demonstrated that both VEGF and bFGF proteins are expressed to different degrees in the human prostatic cancer cell lines. The secretion of VEGF but not bFGF is up-regulated by hypoxia. Linomide is unable to inhibit either basal or hypoxia-induced secretion of VEGF. Linomide also has no effect on secreted bFGF levels. Castration inhibited tumor VEGF but had no effect on bFGF levels in both the androgen-responsive PC-82 and A-2 human prostatic cancers when grown in severe combined immunodeficient mice. When given in combination, castration potentiated the inhibition of tumor growth induced by Linomide alone. This potentiation is not due to a further inhibition in tumor VEGF levels induced by castration. Although both castration and Linomide inhibit angiogenesis, the former accomplishes it by inhibiting VEGF secretion, whereas the latter has multiple effects at several steps in the angiogenic process other than VEGF secretion. Based on their different but complementary mechanisms of action, simultaneous combination of androgen ablation with Linomide enhances the anti-prostatic cancer efficacy compared to either monotherapies alone and warrants testing in humans.

Unusual androgen sensitivity of the androgen-independent Dunning R-3327-G rat prostatic adenocarcinoma: androgen effect on tumor cell loss.

Shessel et al. (Invest. Urol., 17: 529-533, 1980) have reported previously that the serially transplantable Dunning R-3327-G rat prostatic, adenocarcinoma grows faster in intact versus castrated male rats. The present study has demonstrated that this is because the G tumor is composed of androgen-independent but -sensitive prostatic cancer cells. The inclusion that G tumor cells are androgen independent is based upon the observations that these cells are capable of growing following inoculation into castrated male rats and that castration of intact male rats bearing established G tumors induces neither regression of tumor volume nor cessation of the continuous growth of the tumor. The G tumor cells, while being androgen independent, are, however, highly sensitive to androgen for their maximal rate of tumor growth. This androgen sensitivity is demonstrated by the fact that the G tumor cells can be reversibly shifted to a faster or slower growth rate simply by manipulation of the host androgen status. The androgen sensitivity of G tumor growth rate is unusual in that it is not due to androgenic stimulation of cell division but to androgen-induced inhibition of G tumor cell loss (i.e., the rate of G tumor cell loss is reduced by over 50% when androgen is present). The androgen sensitivity of G tumor cell loss is also unusual in that, due to the low level of 5 alpha-reductase activity of the G tumor, the predominant intracellular androgen responsible for this inhibition in untreated intact hosts appears to be testosterone and not dihydrotestosterone (DHT). In castrated rats, however, exogenous treatment with DHT is equally as effective as exogenous testosterone in inhibiting G tumor cell loss. These results suggest that G tumor cells are sensitive to either testosterone or DHT but that in untreated intact hosts little DHT is formed by the tumor cells.

Adaptation versus selection as the mechanism responsible for the relapse of prostatic cancer to androgen ablation therapy as studied in the Dunning R-3327-H adenocarcinoma.

The Dunning R-3327-H rat prostatic adenocarcinoma is a well-differentiated, slow-growing, serially transplantable tumor of spontaneous origin. When intact male rats bearing such an exponentially growing H-tumor s.c. are castrated, tumor growth abruptly stops, demonstrating the initial androgen sensitivity of this tumor. Eventually, however, after an extended period, the tumor invariably relapses and once again appears to grow exponentially. At the time of relapse, the tumor is no longer androgen sensitive but has irreversibly progressed to a completely insensitive state. The mechanism responsible for this irreversible progression has been demonstrated by fluctuation analysis not to be due to environmentally induced adaptation of initially androgen-dependent H-tumor cells to a new androgen-independent state. Instead, the progression is due to the basic heterogeneity of the original H-tumor (i.e., it is composed of a mixture of preexisting clones of both androgen-dependent and androgen-independent tumor cells). Following castration, only the preexisting clones of androgen-independent tumor cells are able to continue exponential growth; the androgen-dependent tumor cells stop proliferating and die. Thus, androgen ablation creates a host environment in which the androgen-independent tumor cells have a highly selective growth advantage over the androgen-dependent cells. Eventually, with time, this selective growth advantage results in a tumor which is completely composed of androgen-independent cells. It is the continuous proliferative growth of these androgen-independent tumor cells which leads to the relapse phenomenon.

Linomide inhibits angiogenesis, growth, metastasis, and macrophage infiltration within rat prostatic cancers.

Linomide, a quinoline-3-carboxamide, has the ability to inhibit the growth of prostatic cancer in vivo but not in vitro (T. Ichikawa et al., Cancer Res., 52: 3022-3028, 1992). The reason for this discrepancy is that linomide inhibits tumor growth not directly but indirectly in vivo via its ability to inhibit the angiogenic response induced within the growing prostatic cancer (J. Vukanovic, et al., Cancer Res., 53: 1833-1837, 1993). Tumor associated macrophages can stimulate angiogenesis via their ability to secrete various cytokines, particularly tumor necrosis factor alpha (TNF-alpha). Treatment of rats with linomide decreases significantly (P < 0.05), by more than 50%, the number of tumor associated macrophages within both locally invasive (i.e., from 20-40 to 10 macrophages/high power field) and highly metastatic primary prostatic cancers (i.e., from 60-70 to 15-37 macrophages/high power field). Monocytes/macrophages isolated from linomide treated rats had a decreased ability to secrete TNF-alpha when challenged in vitro with the bacterial endotoxin, lipopolysaccharide [i.e., 702 +/- 76 (SEM) ng of TNF-alpha/10(5) monocytes/macrophages from control versus 401 +/- 2 ng of TNF-alpha/10(5) monocytes/macrophages from linomide treated rats]. In addition, when rats were treated with linomide and than challenged with lipopolysaccharide in vivo, the resulting elevation in serum TNF-alpha was inhibited by approximately 50% (i.e., 4.56 +/- 1.8 ng/ml of TNF-alpha in control versus 2.9-2.2 ng/ml depending upon the dose of linomide). The ability of linomide to decrease monocyte/macrophage secretion of TNF-alpha is not immediate, however, since the secretion of TNF-alpha induced by lipopolysaccharide challenge of monocytes/macrophages isolated from untreated animals is not decreased by acute (i.e., < 4 h) linomide treatment in vitro. These results demonstrate that the ability of linomide to inhibit the secretion of TNF-alpha by monocytes/macrophages requires either additional time or host factors. To test if natural killer (NK) cells might be one of the additional host factors required for the in vivo abilities of linomide, prostatic cancer bearing rats were treated with appropriate antiserum to deplete NK cells and then tested for their response to linomide treatment. These studies demonstrated that the antitumor, antimetastatic, and antimacrophage effects of linomide were unaffected by NK cell depletion.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of an androgen receptor-null model for identifying the initiation site for androgen stimulation of proliferation and suppression of programmed (apoptotic) death of PC-82 human prostate cancer cells.

Whether androgen regulates the proliferation and survival of androgen-responsive prostate cancer cells directly or indirectly via a paracrine pathway initiated in androgen receptor (AR)-expressing stromal cells is unknown. To resolve this issue, female mice heterozygous for the testicular feminized male loss of function mutation in their X-linked AR genes were cross-bred to T cell-defective homozygous male nude mice. Using a PCR-based restriction enzyme digestion method, the resulting AR/tfm, Nu/nu F1 hybrid females were identified and back-crossed to homozygous male nude mice to produce AR-null male nude mice lacking both AR and T-cell function. Androgen-responsive PC-82 human prostate cancers were xenografted into these AR-null versus AR-wild-type male nude mice. In both backgrounds, the cancer cells did not grow in nonandrogenized hosts. In contrast, PC-82 prostate cancer cells grew with identical characteristics (i.e., take rate, morphology, PSA expression, growth rate, and percentage of cell proliferating or dying) in androgenized hosts of both backgrounds. Likewise, in both backgrounds, androgen ablation of mice bearing growing PC-82 cancers resulted in the inhibition of proliferation and activation of programmed (apoptotic) cell death of the cancer cells. These results demonstrate that both the androgen-stimulated proliferation and the suppression of programmed cell death of PC-82 human prostate cancer cells are initiated by the AR pathway directly within these cancer cells themselves and do not involve initiation by AR-expressing stromal cells in a paracrine manner.

Effectiveness of complete versus partial androgen withdrawal therapy for the treatment of prostatic cancer as studied in the Dunning R-3327 system of rat prostatic adenocarcinomas.

Standard initial therapy for metastatic prostatic cancer involves surgical or chemically induced castration. Castration lowers the serum testosterone level by over 90% but does not completely eliminate all potential serum androgens (i.e., it induces a partial androgen withdrawal). This has led some investigators to suggest that a more complete form of androgen withdrawal in which the very low levels of serum androgens remaining after castration are neutralized by the simultaneous treatment with a direct acting antiandrogen (i.e., complete androgen withdrawal) might be more effective than simply castration alone. To determine whether complete androgen withdrawal is any more effective than partial androgen withdrawal therapy, the slow growing, well differentiated H and the fast growing, poorly differentiated G sublines of the serially transplantable Dunning R-3327 system of rat prostatic adenocarcinomas were used as a test system since both of these cancers are androgen responsive. These studies demonstrated that: (a) complete androgen withdrawal consisting of surgical castration in combination with daily treatment with the potent antiandrogen, cyproterone acetate, was no more effective in terms of tumor growth retardation or overall host survival than was partial androgen withdrawal induced by castration alone; (b) serum testosterone levels must be maintained below 0.5 ng/ml but do not have to be completely eliminated to produce the maximum therapeutic response; and (c) prostatic cancers are more sensitive than is the normal prostate to growth stimulation by serum testosterone.

Human prostatic cancer cells are sensitive to programmed (apoptotic) death induced by the antiangiogenic agent linomide.

Human prostatic cancer cells have a remarkably low rate of proliferation even when they have metastasized to the bone and have become androgen independent (Berges et al., Clin. Cancer Res., 1:473-480, 1995). Due to this low proliferation, patients with such androgen-independent metastatic prostatic cancer cells are rarely treated successfully with the presently available chemotherapeutic agents. Therefore, new approaches are urgently needed which are not dependent on the rate of cancer cell proliferation for their effectiveness. One such approach is to inhibit the angiogenic response within localized and metastatic cancer deposits, since the resultant hypoxia-induced tumor cell death does not require cell proliferation. We have previously demonstrated that the quinoline-3-carboxamide, linomide, is an p.o. active agent which inhibits tumor angiogenesis and thus blood flow in a variety of rat prostatic cancers independent of their growth rate, androgen sensitivity, or metastatic ability. Because of its antiangiogenic effects, linomide treatment induces the hypoxic death of rat prostatic cancer cells, thus inhibiting their net growth and metastases. To determine whether human prostatic cancer cells are similarly sensitive to hypoxia-induced death caused by linomide inhibition of tumor angiogenesis, androgen-independent TSU and PC-3 human prostatic cancer cells were xenotransplanted into SCID mice that were either untreated or treated p.o. with linomide. These studies demonstrated that linomide treatment decreases microvessel density in both androgen-independent human prostatic cancers. Microvessel density was decreased from 1.8 +/- 0.4% of the total area in control tumors to 1.0 +/- 0.2% in linomide-treated TSU tumors [i.e., a 44% decrease in microvessel density (P < 0.05)]. Similarly, a 56% decrease (P < 0.05) was observed in the microvessel density of PC-3 tumors (i.e., 2.7 +/- 0.8% of the area in control tumor versus 1.2 +/- 0.2% in the linomide-treated tumors). This inhibition of angiogenesis increased cell death in both TSU and PC-3 cancer cells. This is reflected in both an increase in the area of necrosis and an increase in the apoptotic index in non-necrotic areas. In untreated TSU tumors, 40 +/- 2% of tumor volume was necrotic. Linomide treatment increased this necrotic percentage to 59 +/- 2% [i.e., 48% increase (P < 0.05)]. Linomide therapy also increased apoptotic cell death in non-necrotic tumor areas. In the untreated TSU tumors, 2.9 +/- 0.6% of tumor cells were apoptotic in the non-necrotic areas, and in the linomide-treated TSU tumors this percentage increased to 3.6 +/- 0.4% [i.e., 24% increase (P < 0.05)].(ABSTRACT TRUNCATED AT 400 WORDS)