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Clinical trials have established the benefit of androgen deprivation therapy (ADT) combined with radiotherapy in prostate cancer. ADT sensitizes prostate cancer to radiotherapy-induced death at least in part through inhibition of DNA repair machinery, but for unknown reasons, adjuvant ADT provides further survival benefits. Here, we show that androgen receptor (AR) expression and activity are durably upregulated following radiotherapy in multiple human prostate cancer models in vitro and in vivo. Moreover, the degree of AR upregulation correlates with survival in vitro and time to tumor progression in animal models. We also provide evidence of AR pathway upregulation, measured by a rise in serum levels of AR-regulated hK2 protein, in nearly 20% of patients after radiotherapy. Furthermore, these men were three-fold more likely to experience subsequent biochemical failure. Collectively, these data demonstrate that radiotherapy can upregulate AR signaling after therapy to an extent that negatively affects disease progression and/or survival., (©2015 American Association for Cancer Research.)

Despite the high incidence and mortality of prostate cancer, the etiology of this disease is not fully understood. In this study, we develop functional evidence for CBP and PTEN interaction in prostate cancer based on findings of their correlate expression in the human disease. Cbp(pc-/-);Pten(pc+/-) mice exhibited higher cell proliferation in the prostate and an early onset of high-grade prostatic intraepithelial neoplasia. Levels of EZH2 methyltransferase were increased along with its Thr350 phosphorylation in both mouse Cbp(-/-); Pten(+/-) and human prostate cancer cells. CBP loss and PTEN deficiency cooperated to trigger a switch from K27-acetylated histone H3 to K27-trimethylated bulk histones in a manner associated with decreased expression of the growth inhibitory EZH2 target genes DAB2IP, p27(KIP1), and p21(CIP1). Conversely, treatment with the histone deacetylase inhibitor panobinostat reversed this switch, in a manner associated with tumor suppression in Cbp(pc-/-);Pten(pc+/-) mice. Our findings show how CBP and PTEN interact to mediate tumor suppression in the prostate, establishing a central role for histone modification in the etiology of prostate cancer and providing a rationale for clinical evaluation of epigenetic-targeted therapy in patients with prostate cancer., (©2014 AACR.)

Overexpression of the histone acetyltransferase p300 is implicated in the proliferation and progression of prostate cancer, but evidence of a causal role is lacking. In this study, we provide genetic evidence that this generic transcriptional coactivator functions as a positive modifier of prostate tumorigenesis. In a mouse model of PTEN deletion-induced prostate cancer, genetic ablation of p300 attenuated expression of the androgen receptor (AR). This finding was confirmed in human prostate cancer cells in which PTEN expression was abolished by RNA interference-mediated attenuation. These results were consistent with clinical evidence that the expression of p300 and AR correlates positively in human prostate cancer specimens. Mechanistically, PTEN inactivation increased AR phosphorylation at serine 81 (Ser81) to promote p300 binding and acetylation of AR, thereby precluding its polyubiquitination and degradation. In support of these findings, in PTEN-deficient prostate cancer in the mouse, we found that p300 was crucial for AR target gene expression. Taken together, our work identifies p300 as a molecular determinant of AR degradation and highlights p300 as a candidate target to manage prostate cancer, especially in cases marked by PTEN loss., (©2014 AACR.)

A major contributor to cancer mortality is recurrence and subsequent metastatic transformation following therapeutic intervention. Therefore, in order to develop new treatment modalities and improve the efficacy of current ones, it is important to understand the molecular mechanisms that promote resistance to therapy in cancer cells. One pathway contributing to therapy resistance is autophagy, a self-digestive process that can eliminate unnecessary or damaged organelles to protect cancer cells from death. We have found that the VEGF-C/NRP-2 axis is involved in the activation of autophagy, which helps cancer cell survival following treatment. Inhibition of mTOR complex 1 activity by this axis is the underlying mechanism for the activation of autophagy. Furthermore, we identified two VEGF-C/NRP-2-regulated genes, LAMP-2 and WDFY-1, that have previously been suggested to participate in autophagy and vesicular trafficking. Upregulation of WDFY-1 following VEGF-C or NRP-2 depletion contributes to cytotoxic drug-mediated cell death. Together, these data suggest a link between the VEGF-C/NRP-2 axis and cancer cell survival despite the presence of chemotherapy-induced stress. Effective targeting of this pathway may lead to the development of new cancer therapies.

The androgen receptor (AR) is the principal target for treatment of non-organ-confined prostate cancer (PCa). Androgen deprivation therapies (ADT) directed against the AR ligand-binding domain do not fully inhibit androgen-dependent signaling critical for PCa progression. Thus, information that could direct the development of more effective ADTs is desired. Systems and bioinformatics approaches suggest that considerable variation exists in the mechanisms by which AR regulates expression of effector genes, pointing to a role for secondary transcription factors. A combination of microarray and in silico analyses led us to identify a 158-gene signature that relies on AR along with the transcription factor SRF (serum response factor), representing less than 6% of androgen-dependent genes. This AR-SRF signature is sufficient to distinguish microdissected benign and malignant prostate samples, and it correlates with the presence of aggressive disease and poor outcome. The AR-SRF signature described here associates more strongly with biochemical failure than other AR target gene signatures of similar size. Furthermore, it is enriched in malignant versus benign prostate tissues, compared with other signatures. To our knowledge, this profile represents the first demonstration of a distinct mechanism of androgen action with clinical relevance in PCa, offering a possible rationale to develop novel and more effective forms of ADT., (©2011 AACR.)

By using LNCaP and its derivative cell lines, we first observed an association between tumor necrosis factor-alpha (TNF-alpha) resistance and hormone independence. Moreover, we found that the expression of tumor necrosis factor receptor-associated death domain (TRADD) was reduced in androgen deprivation-independent cells compared with that in androgen deprivation-dependent cells. TRADD is a crucial transducer for TNF-alpha-induced nuclear factor-kappaB (NF-kappaB) activation. Knocking down TRADD expression in LNCaP cells impaired TNF-alpha-induced NF-kappaB activation and androgen receptor repression, whereas overexpression of TRADD in C4-2B cells restored their sensitivity to TNF-alpha. Finally, we found that androgen deprivation reduces TRADD expression in vitro and in vivo, suggesting that androgen deprivation therapy may promote the development of TNF-alpha resistance by reducing TRADD expression during prostate cancer progression.

ETS variant 1 (ETV1), also known as ETS-related protein 81, is overexpressed in prostate tumors, but whether and how this transcription factor affects tumorigenesis has remained elusive. Here, we show that ETV1 is primarily overexpressed in the most aggressive human prostate tumors. Transgenic ETV1 mice developed prostatic intraepithelial neoplasia as well as hyperplasia/neoplasia in seminal vesicles. Moreover, ETV1 cooperated with the androgen receptor (AR) to bind to the prostate-specific antigen enhancer and stimulate gene transcription. Consistent with its ability to physically interact with AR, ETV1 rendered an ETV1 binding site-driven reporter androgen inducible, and, on the other hand, ETV1 super-induced transcription from an AR binding site on androgen stimulation. In conclusion, our study substantiates that ETV1 overexpression is an underlying cause in the development of prostate and possibly also seminal vesicle cancer. Its interaction with and activation of AR provides a molecular mechanism on how ETV1 exerts its deleterious function. Thus, inhibiting ETV1 or blocking its interaction with AR may represent novel strategies in prostate cancer therapy.

Recurrence and subsequent metastatic transformation of cancer develops from a subset of malignant cells, which show the ability to resist stress and to adopt to a changing microenvironment. These tumor cells have distinctly different growth factor pathways and antiapoptotic responses compared with the vast majority of cancer cells. Long-term therapeutic success can only be achieved by identifying and targeting factors and signaling cascades that help these cells survive during stress. Both microarray and immunohistochemical analysis on human prostate cancer tissue samples have shown an increased expression of vascular endothelial growth factor-C (VEGF-C) in metastatic prostate cancer. We have discovered that VEGF-C acts directly on prostate cancer cells to protect them against oxidative stress. VEGF-C increased the survival of prostate cancer cells during hydrogen peroxide stress by the activation of AKT-1/protein kinase Balpha. This activation was mediated by mammalian target of rapamycin complex-2 and was not observed in the absence of oxidative stress. Finally, the transmembrane nontyrosine kinase receptor neuropilin-2 was found to be essential for the VEGF-C-mediated AKT-1 activation. Indeed, our findings suggest a novel and distinct function of VEGF-C in protecting cancer cells from stress-induced cell death, thereby facilitating cancer recurrence and metastasis. This is distinctly different from the known function of VEGF-C in inducing lymphangiogenesis.

Decreased levels of the prostate-specific homeobox protein NKX3.1 are correlated with hormone-refractory and metastatic prostate cancer. Thus, it is compelling to define the NKX3.1-regulated genes that may be important for the progression of the advanced stage of the disease. In this study, we showed that vascular endothelial growth factor-C (VEGF-C) is one such target gene of NKX3.1. NKX3.1 inhibited VEGF-C expression in prostate cancer, and the loss of NKX3.1 led to increased VEGF-C expression. Histone deacetylase 1 acted as a corepressor of VEGF-C expression along with NKX3.1. Activated RalA acted in synergy with the loss of NKX3.1 for VEGF-C transcription. Patients with deletions at chromosome 8p21.1-p21.2 as a sole deletion developed lymph node metastasis. Interestingly, the higher expression of VEGF-C in prostate cancer is also correlated with lymph node metastasis. Therefore, regulation of VEGF-C expression by NKX3.1 provides a possible mechanism by which the loss of NKX3.1 protein level leads to lymphangiogenesis in the late stages of advanced prostate cancer.

The standard systemic treatment for prostate cancer (PCa) is androgen ablation, which causes tumor regression by inhibiting activity of the androgen receptor (AR). Invariably, PCa recurs with a fatal androgen-refractory phenotype. Importantly, the growth of androgen-refractory PCa remains dependent on the AR through various mechanisms of aberrant AR activation. Here, we studied the 22Rv1 PCa cell line, which was derived from a CWR22 xenograft that relapsed during androgen ablation. Three AR isoforms are expressed in 22Rv1 cells: a full-length version with duplicated exon 3 and two truncated versions lacking the COOH terminal domain (CTD). We found that CTD-truncated AR isoforms are encoded by mRNAs that have a novel exon 2b at their 3' end. Functionally, these AR isoforms are constitutively active and promote the expression of endogenous AR-dependent genes, as well as the proliferation of 22Rv1 cells in a ligand-independent manner. AR mRNAs containing exon 2b and their protein products are expressed in commonly studied PCa cell lines. Moreover, exon 2b-derived species are enriched in xenograft-based models of therapy-resistant PCa. Together, our data describe a simple and effective mechanism by which PCa cells can synthesize a constitutively active AR and thus circumvent androgen ablation.

Androgen receptor (AR) activity is critical for prostate cancer progression. Overexpression of several AR-associated coactivators has been shown to be essential for AR activation during disease progression. The stimuli and signaling pathways leading to overexpression of these coregulators, however, remain largely elusive. Here, we investigated whether androgen signaling, which demarcates critical transitions during prostate cancer disease progression, can affect coregulator expression. We found that expression of four and a half LIM domain protein-2 (FHL2), a key AR coactivator that is overexpressed in prostate cancer and associates with a poor prognosis, is induced strongly by androgens. Androgen induction of this coactivator established a feed-forward mechanism that robustly activated the AR. Stimulation of FHL2 after androgen exposure was time- and dose-dependent and relied on the presence of a functional AR. Androgen induction of FHL2 depended on active transcription of the FHL2 gene, mediated by action of serum response factor (SRF) on its proximal promoter. Loss of SRF, a transcription factor that preferentially regulates the expression of genes involved in mitogenic response and cytoskeletal organization, hampered prostate cancer cell proliferation. These results suggest a novel indirect mechanism of androgen action on FHL2 expression and provide evidence that SRF is an important determinant of AR action in prostate cancer cells.

Systemic prostate cancer therapy requires androgen ablation, which inhibits the production or action of androgens. Prostate cancer ultimately relapses during androgen ablation, and an androgen depletion-independent (ADI) phenotype emerges. Aberrant androgen receptor (AR) activation underlies therapy resistance at this stage of the disease, and mounting evidence implicates the large and highly disordered AR NH2-terminal domain (NTD) as a key mediator of this activity. In this study, we investigated the role of the NTD transactivation unit 5 (TAU5) domain in mediating AR transcriptional activity in cell-based models of prostate cancer progression. AR replacement and Gal4-based promoter tethering experiments revealed that AR TAU5 had a dichotomous function, inhibiting ligand-dependent AR activity in androgen-dependent prostate cancer cells, while enhancing ligand-independent AR activity in ADI prostate cancer cells. Molecular dissection of TAU5 showed that a WxxLF motif was fully responsible for its ligand-independent activity. Mechanistically, WxxLF did not rely on an interaction with the AR ligand-binding domain to mediate ligand-independent AR activity. Rather, WxxLF functioned as an autonomous transactivation domain. These data show that ligand-dependent and ligand-independent AR activation rely on fundamentally distinct mechanisms, and define WxxLF as the major transactivation motif within the AR TAU5 domain.

Standard therapy for nonorgan confined prostate cancer aims to block the production or action of androgens. Although initially successful, antiandrogen therapy eventually fails and androgen depletion independent (ADI) disease emerges. Remarkably, ADI prostate cancers still rely on a functional androgen receptor (AR). Aberrant expression of coregulatory proteins required for the formation of productive AR transcriptional complexes is critical for ADI AR activation. Previously, we have shown that the transcriptional coactivator p300 is required for ADI activation of the AR and is up-regulated in prostate cancer, in which its expression is associated with cell proliferation and predicts aggressive tumor features. The mechanism responsible for the deregulated expression of p300, however, remains elusive. Here, we show that p300 expression in prostate cancer cells is subject to androgen regulation. In several prostate cancer model systems, addition of synthetic and natural androgens led to decreased expression of p300 in a time-dependent and dose-dependent manner. Experiments using AR antagonists or small interfering RNA targeting the AR revealed that down-regulation of p300 depends entirely on the presence of a functional AR. It is noteworthy that androgens down-regulated p300 protein expression while leaving messenger levels unaltered. Conversely, both short-term and long-term androgen deprivation resulted in marked up-regulation of p300 expression. The androgen deprivation-induced increase in p300 expression was not affected by the addition of cytokines or growth factors or by cotreatment with antiandrogens. Moreover, increased p300 expression upon androgen starvation is crucial for prostate cancer cell proliferation, as loss of p300 expression severely reduces expression of cyclins governing G(1)-S and G(2)-M cell cycle transition and decreases 5-bromo-2'-deoxyuridine incorporation.

Germ line mutations in several genes (BRCA1, BRCA2, and CHEK2) whose products are involved in the DNA damage-signaling pathway have been implicated in prostate cancer risk. To identify additional genes in this pathway that might confer susceptibility to this cancer, we analyzed a recently identified DNA damage-response gene, p53AIP1 (a gene encoding for p53-regulated apoptosis-inducing protein 1), for genetic variants in prostate cancer. Five novel germ line variants were identified. The two truncating variants (Ser(32)Stop and Arg(21)insG) were found in 3% (4 of 132) of unselected prostate tumor samples. Genotyping of the two variants in an additional 393 men with sporadic prostate cancer showed a frequency of 3.1% (12 of 393) in contrast to 0.6% (2 of 327) in 327 unaffected men (Fisher's exact test, P = 0.018), with an odds ratio (OR) of 5.1 [95% confidence interval (95% CI), 1.1-23.0]. In addition, two of six tumors carrying the truncating variants were associated with loss of heterozygosity of the wild-type alleles, suggesting that p53AIP1 may act as a tumor suppressor. We also showed that the truncated p53AIP1 was unable to induce apoptosis and suppress cell growth in HeLa and COS-7 cells. These results suggest that loss-of-function variants in p53AIP1 associated with the risk of sporadic prostate cancer and further support the concept that the genetic defects in the DNA damage-response genes play an important role in the development of prostate cancer.

Prostate cancer is the most frequent non-skin cancer in men. Although the mechanisms involved in the progression of prostate cancer are not entirely understood, androgen receptor has been shown to play an important role. Androgen receptor is expressed in both early and late-stage prostate cancer. Also, androgen-regulated pathways are thought to be active as evidenced by elevated levels of prostate-specific antigen (PSA). In addition, several androgen receptor coactivators and cytokines are involved in prostate cancer progression. In this regard, we have shown previously that the coactivator p300 plays a major role in the androgen-independent activation of PSA by interleukin 6 (IL-6), a cytokine involved in late-stage prostate cancer. In this study, we investigated the role of p300 and its homologue CREB-binding protein in prostate cancer cells treated chronically with IL-6. We found that p300 but not CREB-binding protein induced activation of PSA in these cells and that the histone acetyltransferase activity of p300 was critical. This effect was independent of the presence of androgens or antiandrogens. Moreover, we found markedly reduced levels of androgen receptor in these cells and p300 transfection did not affect those levels, suggesting that the p300 effect on PSA could be bypassing the androgen receptor. Transfection with exogenous androgen receptor showed minimal response of PSA to androgens but higher response to p300. We found similar effects of p300 on the androgen response element III, which mediates the androgen receptor-dependent activation of PSA. Finally, we showed that p300 alone regulates expression of the endogenous PSA gene in the IL-6-treated cells. These findings reveal a new insight in the progression of prostate cancer, suggesting that coactivators, such as p300, play more important roles in late-stage prostate cancer, and could regulate androgen-dependent genes in the absence or with very low levels of androgen receptor.

Alterations in nuclear structure distinguish cancer cells from noncancer cells. These nuclear alterations can be translated into quantifiable features by digital image analysis in a process known as quantitative nuclear morphometry. Recently, quantitative nuclear morphometry has been shown to predict metastasis and biochemical recurrence of prostate cancer. However, little is known about the cellular mechanisms underlying these nuclear morphometric changes. Alterations of nuclear matrix proteins are frequently involved in changes of nuclear structure. A number of co-activators interact with these nuclear structure-related proteins, suggesting that they might be involved in quantitative nuclear morphometry changes. We have shown previously that the transcriptional co-activator p300 is involved in prostate cancer progression. However, the ability of a transcriptional regulator like p300 to modulate nuclear morphology has not been described previously. In the present study, we show that p300 expression in prostate cancer biopsy tissue from 95 patients correlates with quantifiable nuclear alterations. Moreover, we show that transfection of p300 into prostate cancer cells in culture induces quantifiable nuclear alterations, such as diameter, perimeter, and absorbance among others, as assessed by digital image analysis. These alterations correlate individually with aggressive features in prostate cancer, such as expression of the proliferation marker Ki-67 and extraprostatic extension of the tumor. Finally, we found that transfection of p300 into prostate cancer cells specifically increases mRNA and protein levels of nuclear matrix peptides lamins A and C, suggesting that these proteins mediate the p300-induced effects. These findings reveal a new insight into the transcriptional and structural regulation of prostate cancer.

Although prostate cancer (PCa) is the most frequently diagnosed cancer in males, little is known about the mechanisms involved in its progression. Recent in vitro studies suggest that coactivators of the androgen receptor play an important role in PCa progression. We have shown previously that p300 is involved in androgen receptor transactivation. In the present work, we studied 95 patients with biopsy-proven PCa who underwent prostatectomy as treatment of their tumors between 1995 and 1998. We found that p300 correlated with in vivo proliferation (P = 0.009) as determined by MIB-I expression. Moreover, high levels of p300 in biopsies predicted larger tumor volumes (P < 0.001), extraprostatic extension (P = 0.003), and seminal vesicle involvement (P = 0.002) at prostatectomy, as well as PCa progression after surgery (P = 0.01). Furthermore, we found that the disruption of p300 transcripts through small interfering RNA inhibited PCa cell proliferation both at the basal level and on interleukin 6 stimulation. We conclude that p300 plays an important role in PCa cell proliferation, as well as PCa progression.

Prostate cancer (PCa) begins as an androgen-dependent tumor that will eventually progress to an androgen-independent stage after androgen ablation. Although little is understood about this transition to androgen independence, the androgen receptor (AR) appears to be involved. The coactivator p300 has been shown to interact with the AR during its androgen-dependent transactivation. We show that p300 is involved downstream of the mitogen-activated protein kinase pathway during transactivation of the AR by interleukin-6 (IL-6). Furthermore, we demonstrate that sequestration of p300 with E1A inhibits the IL-6-dependent transactivation of the AR, and that increasing amounts of p300 reverse this inhibition. A mutant p300 that lacks histone acetyltransferase (HAT) activity did not reverse E1A-mediated inhibition. By using small-interference RNA designed to target p300 transcripts, we demonstrate that, after silencing p300, there was no induction of AR activity by IL-6. These findings reveal a unique role for p300 and its HAT activity, indicating that it is necessary for the ligand-independent transactivation of the AR in androgen-independent PCa cells.

Prostate cancer cells depend on androgens and the androgen receptor (AR) for survival. However, after androgen ablation therapy, tumors relapse to an androgen-refractory state. To determine whether the androgen receptor is critical for proliferation of androgen-refractory prostate cancer cells, we disrupted the activity of the androgen receptor with an antibody and an AR mRNA hammerhead ribozyme in the following cell lines: LNCaP (androgen-sensitive), LNCaP-Rf and LNCaP-C4 (androgen-refractory), and DU-145 (androgen-insensitive). Microinjection of either antibody or ribozyme inhibited proliferation of androgen-refractory cells. These findings demonstrate that the AR is critical for proliferation of androgen-refractory cells, even in the absence of androgens.

Prostate cancer (PCa) progression is aided by abnormal autocrine growth factor loops. We screened for small cell-permeable inhibitors of receptor tyrosine kinases that could block their signaling and trigger cell death in PCa cell lines. We found that the human epidermal growth factor receptor (HER)-2/neu inhibitor tyrphostin AG825 is preferentially toxic to PCa cells that are phenotypically androgen independent. These effects were dose and time dependent in the human LNCaP, C4, and C4-2 cell line models of progression and correlated with the inhibition of HER-2/neu phosphoactivation and its down-regulation. In addition, we show that the inhibition of HER-2/neu signaling with AG825 triggers an imbalance between extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase activation, which leads to p38-dependent apoptosis. Inhibition of HER-1 with Compound 56 had no effect. These findings suggest that the androgen-independent C4 and C4-2 cells can be killed by selectively inhibiting their HER-2/neu signaling pathway and provide insights into the mechanism of action of AG825 in PCa cells.

We developed a highly sensitive splice variant-specific reverse transcriptase-PCR (RT-PCR) assay for human glandular kallikrein (hK2) mRNA and tested its ability to detect metastatic disease in men with clinically localized prostate cancer. An RT-PCR assay using primers spanning intron IV and including a significant portion of the 3' untranslated region of the hKLK2 gene, with maximum nonhomology to both hK1 and hK3, was developed. The limit of detection of the assay was five copies of hK2 cDNA and one LNCaP cell in 10(9) lymphoblasts. RT-PCR-hK2 was performed on preoperative peripheral blood specimens from 228 consecutive radical prostatectomy patients as well as 7 metastatic prostate cancer patients and 14 healthy men without prostate cancer. This new RT-PCR-hK2 assay amplifies two distinct fragments. The larger fragment (hK2-U) is approximately 680 bp in length and corresponds to the amplified product of a previously reported splice variant in the splice donor site of intron IV in the hKLK2 gene. The smaller fragment (hK2-L) is approximately 643 bp in length and corresponds to the amplified product of the native hK2 mRNA. Whereas the RT-PCR-hK2-L assay was positive in 71% of our patients with metastatic prostate cancer, 14% of healthy control men also tested positive. By univariate (P = 0.028) and multivariate (P = 0.0269) analysis, which controlled for preoperative PSA, clinical stage, and biopsy Gleason score, RT-PCR-hK2-L status added prognostic information to the prediction of lymph node-positive disease. We have developed a new RT-PCR assay which demonstrates a high sensitivity for detecting hK2 mRNA. Preoperative RT-PCR-hK2-L status helps predict pathological lymph node positivity in patients with clinically localized prostate cancer.

Causing prostate cancer cells to express functionally active sodium iodide symporter (NIS) by targeted NIS gene transfer might offer the possibility of radioiodine therapy of prostate cancer. Therefore, we investigated radioiodine accumulation and therapeutic effectiveness of 131I in NIS-transfected prostate cancer cells in vitro and in vivo. The human prostatic adenocarcinoma cell line LNCaP was stably transfected with NIS cDNA under the control of the prostate-specific antigen promoter. The stably transfected LNCaP cell line NP-1 showed perchlorate-sensitive, androgen-dependent iodide uptake in vitro that resulted in selective killing of these cells by 131I in an in vitro clonogenic assay. Xenografts were established in athymic nude mice and imaged using a gamma camera after i.p. injection of 500 microCi of 123I. In contrast to the NIS-negative control tumors (P-1) which showed no in vivo uptake of 123I, NP-1 tumors accumulated 25-30% of the total 123I administered with a biological half-life of 45 h. In addition, NIS protein expression in LNCaP cell xenografts was confirmed by Western blot analysis and immunohistochemistry. After a single i.p. application of a therapeutic 131I dose (3 mCi), significant tumor reduction was achieved in NP-1 tumors in the therapy group compared with P-1 tumors and tumors in the control group. In conclusion, a therapeutic effect of 131I has been demonstrated in prostate cancer cells after induction of tissue-specific iodide uptake activity by prostate-specific antigen promoter-directed NIS expression in vitro and in vivo. This study demonstrates the potential of NIS as a novel therapeutic gene for nonthyroidal cancers, in particular prostate cancer.

Human kallikrein (hK) 2 is an arginine-selective serine protease expressed predominantly in the prostate that has an 80% sequence identity with prostate-specific antigen. Expression of hK2 is elevated in the tumor epithelium compared to benign prostate tissue. We have purified, sequenced, and identified a novel hK2 complex in prostate tissue consisting of hK2 and a serine protease inhibitor known as protease inhibitor-6 (PI-6). This 64-kDa SDS-PAGE stable complex is elevated in the tumor and is approximately 10% of total hK2. No comparable complex of prostate-specific antigen was detected. PI-6, also known as cytoplasmic antiprotease, has been characterized as an intracellular inhibitor of trypsin and chymotrypsin-like proteases, which has high homology to plasminogen activator inhibitor 1 and 2. The physiological role of PI-6 in the prostate and its relationship to hK2 and prostate cancer are under investigation.

Currently, no curative therapy for metastatic prostate cancer exists. Causing prostate cancer cells to express functionally active sodium iodide symporter (NIS) would enable those cells to concentrate iodide from plasma and might offer the ability to treat prostate cancer with radioiodine. Therefore, the aim of our study was to achieve tissue-specific expression of full-length human NIS (hNIS) cDNA in the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP and in subcell lines C4, C4-2, and C4-2b in vitro. For this purpose, an expression vector was generated in which full-length hNIS cDNA coupled to the prostate-specific antigen (PSA) promoter has been ligated into the pEGFP-1 vector (NIS/PSA-pEGFP-1). The PSA promoter is responsible for androgen-dependent expression of PSA in benign and malignant prostate cells and was therefore used to mediate androgen-dependent prostate-specific expression of NIS. In addition, two control vectors were designed, which consist of the pEGFP-1 vector containing the PSA promoter without NIS cDNA (PSA-pEGFP-1) and NIS cDNA without the PSA promoter (NIS-pEGFP-1). Prostate cancer cells were transiently transfected with each of the above-described expression vectors, incubated with or without androgen (mibolerone) for 48 h, and monitored for iodide uptake activity. In addition, stably transfected LNCaP cell lines were established for each vector. Prostate cells transfected with NIS/PSA-pEGFP-1 showed perchlorate-sensitive, androgen-dependent iodide uptake in a range comparable to that observed in control cell lines transfected with hNIS cDNA. Perchlorate-sensitive iodide uptake was not observed in cells transfected with NIS/PSA-pEGFP-1 and treated without androgen or in cells transfected with the control vectors. In addition, prostate cancer cell lines without PSA expression (PC-3 and DU-145) did not show iodide uptake activity when transfected with NIS/PSA-pEGFP-1. Western blotting of LNCaP and C4-2b cell membranes transfected with NIS/PSA-pEGFP-1 using a monoclonal antibody that recognizes the COOH-terminus of hNIS revealed a band with a molecular weight of 90,000 that was not detected in androgen-deprived cells or in cells transfected with the control vectors, as well as a minor band at Mr 150,000 in transiently transfected LNCaP cell membranes. In conclusion, tissue-specific androgen-dependent iodide uptake activity has been induced in prostate cancer cells by PSA promoter-directed NIS expression. This study represents an initial step toward therapy of prostate cancer with radioiodine.

p73, a first p53 relative, has recently been identified and demonstrated to be monoallelically expressed. This protein shows significant amino acid sequence and functional similarities to p53. However, it is unclear whether this protein functions as a tumor suppressor. To elucidate the role of p73 in tumor development, we investigated the expression of the p73 gene in lung cancer. In a comparison between normal lung and tumor tissues, p73 was more highly expressed in tumors. Moreover, using a C/T polymorphism in exon 2 for allele-specific expression analysis in 21 pairs of lung tumors and matched normal tissues, we found that five heterozygous samples exclusively expressed both alleles in tumors while showing monoallelic expression in matched normal tissues. This result was confirmed by single-nucleotide primer extension analysis. Mutation analysis of all 13 coding exons of the gene in 21 lung tumor DNAs revealed several polymorphisms, but no tumor-specific mutations were detected. These findings strongly suggest that p73 may play an important role in lung tumorigenesis through activation of a silent allele and overexpression of wild-type p73 rather than as a tumor suppressor.

Early growth-response (EGR) genes are nuclear transcription factors that are implicated in regulating cell proliferation. Because these genes show divergent expression in various human tumors, we sought to determine their expression in nonmalignant and malignant prostate tissues. Total RNA extracted from prostate tissues was probed with EGR-1, EGR-2, and EGR-alpha cDNA for Northern blots and digoxigenin-labeled cRNA for in situ hybridization. Both Northern blot and in situ hybridization analyses demonstrated increased EGR-1, but not EGR-2 or EGR-alpha expression, in malignant prostate tissue as compared with weak expression in nonmalignant tissue. EGR-1 mRNA was quantified in 96 prostate specimens (86 adenocarcinomas representing different Gleason scores and 10 benign tissues showing no histological manifestation of benign prostatic hypertrophy) using in situ hybridization with an 35S-labeled cRNA probe. EGR-1 mRNA was expressed at significantly higher levels in cancer than in normal prostate (P < 0.001). In cancer with Gleason scores 8-10, the expression of EGR-1 was higher compared with those of lower Gleason scores (P < 0.005). Immunohistochemical staining showed predominately basal cell nuclear EGR-1 protein in prostatic acini. Nuclear staining was weak in nonmalignant tissues, more intense in moderately differentiated carcinoma, and most intense in poorly differentiated carcinoma. These results show that EGR-1 is overexpressed in prostate cancer and suggest a role for EGR-1 in prostate cancer growth.

Human glandular kallikrein (hK2) protein, like prostate-specific antigen (PSA), is produced mainly in prostatic epithelium. It may be useful as a new diagnostic indicator for prostate cancer. Recently, a number of hK2-specific monoclonal antibodies have been developed that enable us to detect hK2 protein in human prostate tissue, seminal fluid, and sera. Whether hK2 can be expressed, like PSA, in nonprostatic cells is not known. In this study, we have characterized the presence of hK2 in an androgen-responsive breast cancer cell line T47-D at both the protein and mRNA levels with an immunoassay, Western blot analysis, Northern blot analysis, and the reverse transcription-PCR. Using a sensitive immunoassay with monoclonal antibodies to hK2, we found that T47-D cells could be induced with androgens, mineralocorticoids, glucocorticoids, and progestins to produce significantly more hK2 than PSA. Estrogens failed to mimic the effect of the other steroids, blocking instead the stimulatory effect of androgens. Androgen induction of hK2 in T47-D cells was dose dependent. More interestingly, we found that the hK2 in androgen-induced T47-D cell spent media appears to be the pro-form of hK2 rather than mature hK2. Our study demonstrates that hK2, a serine protease thought to be found only in prostate-related tissues and fluids, is also produced in a breast cancer cell line T47-D after steroid stimulation. This finding suggests that hK2 may have a potential role in breast cancer as well as prostatic cancer and will be the impetus for further studies of hK2 distribution and function.

Proliferating cell nuclear antigen (PCNA) expression is required for DNA replication. Because androgens are critical for prostate cell proliferation, we investigated the effects of androgen on PCNA expression in the prostatic cancer cell line LNCaP. Flow cytometric analysis was used to measure cellular DNA content with dual labeling of PCNA. Semiconfluent LNCaP cells were grown in serum-free medium containing varying concentrations of the synthetic androgen mibolerone and processed for either fluorescence-activated cell sorting or Western analysis. Supplementation of serum-free medium with androgens resulted in dose-dependent changes in PCNA immunoreactivity, with maximum stimulation (2-fold) being achieved at 48 h with 10(-9)M mibolerone. Non-androgenic steroids did not change PCNA immunoreactivity compared with untreated controls, and the antiandrogen, casodex, inhibited the mibolerone-stimulated increase in PCNA immunoreactivity, suggesting that the androgenic induction of PCNA is mediated through the androgen receptor. The presence of a non-consensus androgen response element in the promoter region of the PCNA gene led us to investigate wether androgen responsiveness of the PCNA gene in LNCaP cells might be mediated at the transcriptional level. No change in steady-state mRNA for PCNA with androgen administration was observed. However, an investigation of the androgenic regulation of PCNA protein stability indicated that androgen treatment increased the half-life of 35S-labeled PCNA protein. In addition, polysome run-off translation assays demonstrated an increase in PCNA protein after a 6-h stimulation of LNCaP cells with 10(-9)M mibolerone. These data suggest that androgen induction of prostate cell proliferation may be mediated, at least in part, through PCNA at the posttranscriptional level.

Prostate-specific antigen (PSA) is the most sensitive marker available for monitoring the progression of prostate cancer and response to therapy. In a previous study, we demonstrated tissue-specific expression of PSA glycoprotein and mRNA and its regulation through the androgen receptor. In this study, we examine the effects of protein kinase A (PKA) and protein kinase C (PKC) on the androgen regulation of PSA in a human adenocarcinoma cell line, LNCaP. Northern blot analysis demonstrated that forskolin, an activator of PKA, had no effect on the androgen regulation of PSA. However, the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a direct activator of PKC, showed a time- and dose-dependent repression of the androgen regulation of PSA glycoprotein and mRNA. The biologically inactive phorbol ester, 4 alpha-phorbol-12,13-didecanoate, had no effect. Staurosporine, a PKC inhibitor, blocked the TPA-mediated repression of the androgenic stimulation of PSA glycoprotein. In addition, the calcium ionophore, A23187, was able to simulate the actions of TPA, presumably through activation of PKC via calcium mobilization. In summary, the androgenic regulation of PSA protein and mRNA is repressed by tumor-promoting phorbol esters through the PKC pathway. This indicates that the effects of TPA may be secondary to repressed gene transcription or altered mRNA stability. In addition, this study emphasizes that the androgenic regulation of PSA is complex and may involve other extracellular transduction signals.

Prostate-specific antigen (PSA) is a member of the kallikrein gene family and is expressed exclusively in human prostatic epithelial cells. PSA protein has been an important biological marker for prostate cancers. Until now, very little was known about the regulation of PSA expression in prostatic cells. In this study, we have developed a specific oligonucleotide probe which recognizes PSA but not the human glandular kallikrein. This is crucial because both PSA and human glandular kallikrein are expressed in the prostate at relatively high levels and have high nucleotide sequence homology (greater than 82%). Utilizing a S-labeled PSA-specific probe, PSA mRNA was localized within the glandular epithelium of the prostate. Northern blot analysis detected a single 1.6-kilobase transcript in LNCaP cells, a cell line derived from a human prostate adenocarcinoma metastasis. Therefore, LNCaP cells were used to study the androgenic effects on PSA mRNA expression. A time course study demonstrated that PSA mRNA was induced by mibolerone (a nonmetabolizable synthetic androgen) and reached maximal levels after 9 h. The induction of PSA mRNA required as little as 0.3 nM mibolerone. In addition to mibolerone, PSA mRNA could be induced by the natural androgen, dihydrotestosterone, but not by the synthetic glucocorticoid, dexamethasone, or the synthetic estrogen, diethylstilbestrol. Moreover, in the presence of dihydrotestosterone, PSA mRNA was depressed by hydroxyflutamide (an antiandrogen). These results suggest strongly that the androgenic effects on PSA mRNA in LNCaP cells may be via the function of the androgen receptor.

In vitro studies were conducted to determine whether conditioned medium from rat fetal urogenital sinus explants would affect phenotypic characteristics of NBT-II urinary bladder carcinoma cells in culture. NBT-II cells were exposed to medium (30%, v/v) conditioned for 48 h by intact urogenital sinus explants derived from 18-day fetal rats. Upon exposure for 23 h the [3H]thymidine incorporation by NBT-II cells was decreased by 40.3% relative to control cultures. This effect was paralleled by a similar decrease in proliferation. NBT-II cultures decreased in cell number by 32.1 and 45.8% on days 2 and 4, respectively, after exposure to conditioned medium. Although cell proliferation was inhibited, conditioned medium acted to induce an increase in protein secretion. An increase of 18.6% was observed in the incorporation of [35S]methionine into newly synthesized, secreted proteins by NBT-II cells exposed to conditioned medium for 23 h. Morphologically the NBT-II cells exposed to conditioned medium were larger, more spread out, and exhibited a greater array of lamellipodia and filopodia, although [35S]methionine incorporation into cellular proteins was decreased by 11.1%. These results suggest that diffusable factors produced by fetal urogenital sinus explants can induce changes in proliferation, protein synthesis, protein secretion, and phenotypic morphology of NBT-II carcinoma cells in culture.