Growth factors in human prostate cancer cells: implications for an improved treatment of prostate cancer

It has been previously shown that estrogens may exert their action on human breast cancer cells through coordinated control of secreted growth factors which act in an autocrine and paracrine fashion. Growth stimulation of the androgen receptor negative prostate carcinoma cell line DU-145 by dihydrotestosterone in the presence of the androgen-responsive human prostate carcinoma cell line LNCaP now indicates that androgens may regulate growth of prostate carcinoma cells through related mechanisms. A variety of androgen-regulated growth modulatory activities with autocrine and paracrine potential can be detected in conditioned media from LNCaP cells partially purified by ion exchange chromatography. Androgen-induced growth of LNCaP cells is partially inhibited by the polyanions suramin and dextran sulfates which antagonize growth factor action. These data suggest the existence of at least two different mechanisms of growth regulation by androgen which can be distinguished by their different sensitivity to growth factor inhibitory agents. We conclude that the combination of antipeptidergic substances and androgen withdrawal would represent a new and promising strategy for treatment of human prostate cancer.     

Novel non-steroidal/non-anilide type androgen antagonists with an isoxazolone moiety

3-Substituted (Z)-4-(4-N,N-dialkylaminophenylmethylene)-5(4H)-isoxazolones and related compounds were designed and prepared as candidates for structurally novel androgen antagonists. Several compounds showed potent anti-androgenic activity as assessed by nuclear androgen receptor binding assay and growth inhibition assay using androgen-dependent Shionogi carcinoma cells SC-3. They were approximately 10--220 times more potent than flutamide in these assay systems. They also showed anti-androgenic activity toward prostate tumor cell line LNCaP, which has an aberrant nuclear androgen receptor.     

Permanent phenotypic and genotypic changes of prostate cancer cells cultured in a three-dimensional rotating-wall vessel

A three-dimensional (3D) integrated rotating-wall vessel cell-culture system was used to evaluate the interaction between a human prostate cancer cell line, LNCaP, and microcarrier beads alone, or microcarrier beads previously seeded with either prostate or bone stromal cells. Upon coculture of LNCaP cells with microcarrier beads either in the presence or in the absence of prostate or bone stromal cells, 3D prostate organoids were formed with the expected hormonal responsiveness to androgen, increased cell growth, and prostate-specific antigen production. In this communication, we define permanent phenotypic and genotypic changes of LNCaP cells upon coculture with microcarrier beads alone, or with microcarrier beads previously seeded with either prostate or bone stromal cells. Most notably, we observed selective genetic changes, i.e., chromosomal losses or gains, as evaluated by both conventional cytogenetic and comparative genomic hybridization, in LNCaP sublines derived from the prostate organoids. Moreover, the derivative LNCaP cells appear to have altered growth profiles, and exhibit permanent and stable changes in response to androgen, estrogen, and growth factors. The derivative LNCaP sublines showed increased anchorage-independent growth rate, and enhanced tumorigenicity and metastatic potential when inoculated orthotopically in castrated athymic mice. Our results support the hypothesis that further nonrandom genetic and phenotypic changes in prostate cancer epithelial cells can occur through an event that resembles "adaptive mutation" such as has been described in bacteria subjected to nutritional starvation. The occurrence of such permanent changes may be highly contact dependent, and appears to be driven by specific microenvironmental factors surrounding the tumor cell epithelium grown as 3D prostate organoids.     

Epidermal growth factor receptor-dependent stimulation of amphiregulin expression in androgen-stimulated human prostate cancer cells.

Amphiregulin is a heparin-binding epidermal growth factor (EGF)-related peptide that binds to the EGF receptor (EGF-R) with high affinity. In this study, we report a role for amphiregulin in androgen-stimulated regulation of prostate cancer cell growth. Androgen is known to enhance EGF-R expression in the androgen-sensitive LNCaP human prostate carcinoma cell line, and it has been suggested that androgenic stimuli may regulate proliferation, in part, through autocrine mechanisms involving the EGF-R. In this study, we demonstrate that LNCaP cells express amphiregulin mRNA and peptide and that this expression is elevated by androgenic stimulation. We also show that ligand-dependent EGF-R stimulation induces amphiregulin expression and that androgenic effects on amphiregulin synthesis are mediated through this EGF-R pathway. Parallel studies using the estrogen-responsive breast carcinoma cell line, MCF-7, suggest that regulation of amphiregulin by estrogen may also be mediated via an EGF-R pathway. In addition, heparin treatment of LNCaP cells inhibits androgen-stimulated cell growth further suggesting that amphiregulin can mediate androgen-stimulated LNCaP proliferation. Together, these results implicate an androgen-regulated autocrine loop composed of amphiregulin and its receptor in prostate cancer cell growth and suggest that the mechanism of steroid hormone regulation of amphiregulin synthesis may occur through androgen upregulation of the EGF-R and subsequent receptor-dependent pathways.     

Androgen inhibits the growth of carcinoma cell lines established from prostate cancer xenografts that escape androgen treatment

Most prostate cancers escape endocrine therapy by diverse mechanisms. One of them might be growth repression by androgen. We reported that androgen represses the growth in culture of MOP cells (a sub-line of LNCaP cells) and that of MOP cell xenografts, although tumor growth becomes androgen-independent (AI). Here we explore whether AI tumors contain androgen-responsive cells. ME carcinoma cells were established from AI tumors. The responses to androgen were examined by cell counting, DAPI labeling, flow cytometry, PSA immunoassay and tumor size follow-up. Androgen receptors (AR) were analyzed by western blotting and DNA sequencing. The pattern of responses of these cells to androgen was compared to that of MOP cells and that of JAC cells established from LNCaP-like MOP cells. R1881, a synthetic androgen: (1) repressed the growth of all the six ME cell lines obtained, MOP and JAC cells, (2) augmented the secretion of PSA, (3) induced spectacular cell bubbling/fragmentation and (4) blocked the cell cycle and induced a modest increase of apoptosis. All the androgen-repressed cells expressed the same level of mutated AR as LNCaP cells. In nude mice, the growth of ME-2 cell xenografts displayed transient androgen repression similar to that of MOP cells. In culture neither fibroblasts nor extra-cellular matrix altered the effects of R1881 on cell proliferation. These results demonstrate that androgen-independent tumors contain androgen-responsive cells. The apparent discrepancy between the responses to androgen of tumors and those of carcinoma cells in culture suggests that microenvironmental factors contribute to the androgen responsiveness of tumor cells in vivo. These modifications, albeit unspecified, could be suitable targets for restoring the androgen responsiveness of AI tumors.     

Sex steroid hormone metabolism and prostate cancer

The growth and function of the prostate is dependent on androgens. The two predominant androgens are testosterone, which is formed in the testis from androstenedione and 5alpha-dihydrotestosterone, which is formed from testosterone by 5alpha-reductases and is the most active androgen in the prostate. Prostate cancer is one of the most common cancers among men and androgens are involved in controlling the growth of androgen-sensitive malignant prostatic cells. The endocrine therapy used to treat prostate cancer aims to eliminate androgenic activity from the prostatic tissue. Most prostate cancers are initially responsive to androgen withdrawal but become later refractory to the therapy and begin to grow androgen-independently. Using LNCaP prostate cancer cell line we have developed a cell model to study the progression of prostate cancer. In the model androgen-sensitive LNCaP cells are transformed in culture conditions into more aggressive, androgen-independent cells. The model was used to study androgen and estrogen metabolism during the transformation process. Our results indicate that substantial changes in androgen and estrogen metabolism occur in the cells during the process. A remarkable decrease in the oxidative 17beta-hydroxysteroid dehydrogenase activity was seen whereas the reductive activity seemed to increase. The changes suggest that during transformation estrogen influence is increasing in the cells. This is supported by the cDNA microarray screening results which showed over-expression of several genes up-regulated by estrogens in the LNCaP cells line representing progressive prostate cancer. Since local steroid metabolism controls the bioavailability of active steroid hormones in the prostate, the variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of the organ.     

KLF9 suppresses cell growth and induces apoptosis via the AR pathway in androgen-dependent prostate cancer cells

Kruppel-like factors (KLFs) play an important role in many biological processes including cell proliferation, differentiation and development. Our study showed that the level of KLF9 is lower in PCa cell lines compared to a benign prostate cell line; the androgen-independent cell line PC3 expresses significantly lower KLF9 than the androgen-dependent cell line, LNCaP. Forced overexpression of KLF9 suppressed cell growth, colony formation, and induced cell apoptosis in LNCaP cells. We also found that KLF9 expression was induced in response to apoptosis caused by flutamide, and further addition of dihydrotestosterone antagonized the action of flutamide and significantly decreased KLF9 expression. Furthermore, activation of the androgen receptor (AR) was inhibited by the overexpression of KLF9. Our research shows that KLF9 is lower in androgen-independent cell lines than in androgen-dependent cell lines; Overexpression of KLF9 dramatically suppresses the proliferation, anchorage-independent growth, and induces apoptosis in androgen-dependent cells; KLF9 inhibition on prostate cancer cell growth may be acting through the AR pathway. Our results therefore suggest that KLF9 may play a significant role in the transition from androgen-dependent to androgen-independent prostate cancer and is a potential target of prevention and therapy.     

Neuropeptide-Induced Androgen Independence in Prostate Cancer Cells: Roles of Nonreceptor Tyrosine Kinases Etk/Bmx, Src, and Focal Adhesion Kinase

The bombesin/gastrin-releasing peptide (GRP) family of neuropeptides has been implicated in various in vitro and in vivo models of human malignancies including prostate cancers. It was previously shown that bombesin and/or neurotensin (NT) acts as a survival and migratory factor(s) for androgen-independent prostate cancers. However, a role in the transition from an androgen-dependent to -refractory state has not been addressed. In this study, we investigate the biological effects and signal pathways of bombesin and NT on LNCaP, a prostate cancer cell line which requires androgen for growth. We show that both neurotrophic factors can induce LNCaP growth in the absence of androgen. Concurrent transactivation of reporter genes driven by the prostate-specific antigen promoter or a promoter carrying an androgen-responsive element (ARE) indicate that growth stimulation is accompanied by androgen receptor (AR) activation. Furthermore, neurotrophic factor-induced gene activation was also present in PC3 cells transfected with the AR but not in the parental line which lacks the AR. Given that bombesin does not directly bind to the AR and is known to engage a G-protein-coupled receptor, we investigated downstream signaling events that could possibly interact with the AR pathway. We found that three nonreceptor tyrosine kinases, focal adhesion kinase (FAK), Src, and Etk/BMX play important parts in this process. Etk/Bmx activation requires FAK and Src and is critical for neurotrophic factor-induced growth, as LNCaP cells transfected with a dominant-negative Etk/BMX fail to respond to bombesin. Etk's activation requires FAK, Src, but not phosphatidylinositol 3-kinase. Likewise, bombesin-induced AR activation is inhibited by the dominant-negative mutant of either Src or FAK. Thus, in addition to defining a new G-protein pathway, this report makes the following points regarding prostate cancer. (i) Neurotrophic factors can activate the AR, thus circumventing the normal growth inhibition caused by androgen ablation. (ii) Tyrosine kinases are involved in neurotrophic factor-mediated AR activation and, as such, may serve as targets of future therapeutics, to be used in conjunction with current antihormone and antineuropeptide therapies.     

Restoration of transforming growth factor-beta type II receptor reduces tumorigenicity in the human adrenocortical carcinoma SW-13 cell line.

Transforming growth factor-beta (TGF-beta) is a potent growth suppressor. Acquisition of TGF-beta resistance has been reported in many tumors, and has been associated with reduced TGF-beta receptor expression. In this study, we examined TGF-beta 1, TGF-beta type I receptor (TbetaRI) and TGF-beta type II receptor (TbetaRII) expression in SW-13 adrenocortical carcinoma cells by Northern and Western blot analysis. SW-13 cells did not express TbetaRII mRNA or protein. We have investigated the role of TbetaRII in modulating tumorigenic potential using stably transfected SW-13 cells with TbetaRII expression plasmid. TbetaRII-positive SW-13 cell growth was inhibited by exogenous human TGF-beta1 (hTGF-beta1) in a dose-dependent manner. In contrast, SW-13 cells and control clones transfected with empty vector remained hTGF-beta1-insensitive. Xenograft examination in athymic nude mice demonstrated that TbetaRII-positive SW-13 cells reduced tumor-forming activity. Reconstructing the TbetaRII can lead to reversion of the malignant phenotype of TbetaRII-negative human adrenocortical carcinoma, which contains SW-13 cells. Reduced TbetaRII expression may play a critical role in determining the malignant phenotype of human adrenocortical carcinoma.     

Product of aromatase activity in intact LNCaP and MCF-7 human cancer cells

We investigated conversion rates of androgens to estrogens in cultured, hormone-responsive prostate (LNCaP) and breast (MCF-7) human cancer cells. For this purpose, we adopted an intact cell analysis, whereby cells were incubated for different incubation times in the presence of close-to-physiological (1 nM) or supraphysiological (1 μM) concentrations of labelled androgen precursors, i.e. testosterone (T) and androstenedione (Δ⁴Ad). The aromatase activity, as measured by estrogen formation, was detected in LNCaP cells (0.5 pmol/ml), even though to a significantly lower extent than in MCF-7 cells (5.4 pmol/ml), using 1 μM T after 72 h incubation. Surprisingly, LNCaP cells displayed a much higher aromatase activity when T was used as a substrate with respect to Δ⁴Ad. In either cell line, T transformation to Δ⁴Ad was relatively low, attaining only 2.8% in LNCaP and 7.5% MCF-7 cells. However, T was mostly converted to conjugates (over 95%), glucuronides and some sulphates, in LNCaP cells, whereas it was only partly converted to sulphates (<10%) in MCF-7 cells. Aromatase activity seems to be inconsistent in LNCaP cells, being strongly affected by culture conditions, especially by fetal calf serum (FCS). Further studies should assess the regulation of aromatase expression by serum or growth factors in different human cancer cells, also using anti-aromatase and/or anti-estrogen compounds, in different culture conditions.     

Antioxidants Abrogate Alpha-Tocopherylquinone-Mediated Down-Regulation of the Androgen Receptor in Androgen-Responsive Prostate Cancer Cells

Tocopherylquinone (TQ), the oxidation product of alpha-tocopherol (AT), is a bioactive molecule with distinct properties from AT. In this study, AT and TQ are investigated for their comparative effects on growth and androgenic activity in prostate cancer cells. TQ potently inhibited the growth of androgen-responsive prostate cancer cell lines (e.g., LAPC4 and LNCaP cells), whereas the growth of androgen-independent prostate cancer cells (e.g., DU145 cells) was not affected by TQ. Due to the growth inhibitory effects induced by TQ on androgen-responsive cells, the anti-androgenic properties of TQ were examined. TQ inhibited the androgen-induced activation of an androgen-responsive reporter and inhibited the release of prostate specific antigen from LNCaP cells. TQ pretreatment was also found to inhibit AR activation as measured using the Multifunctional Androgen Receptor Screening assay. Furthermore, TQ decreased androgen-responsive gene expression, including TM4SF1, KLK2, and PSA over 5-fold, whereas AT did not affect the expression of androgen-responsive genes. Of importance, the antiandrogenic effects of TQ on prostate cancer cells were found to result from androgen receptor protein down-regulation produced by TQ that was not observed with AT treatment. Moreover, none of the androgenic endpoints assessed were affected by AT. The down-regulation of androgen receptor protein by TQ was abrogated by co-treatment with antioxidants. Overall, the biological actions of TQ were found to be distinct from AT, where TQ was found to be a potent inhibitor of cell growth and androgenic activity in androgen-responsive prostate cancer cells.     

The androgen receptor: Functional structure and expression in transplanted human prostate tumors and prostate tumor cell lines

The growth of the majority of prostate tumors is androgen-dependent, for which the presence of a functional androgen receptor is a prerequisite. Tumor growth can be inhibited by blockade of androgen receptor action. However, this inhibition is transient. To study the role of the androgen receptor in androgen-dependent and androgen-independent prostate tumor cell growth, androgen receptor mRNA expression was monitored in six different human prostate tumor cell lines and tumors, which were grown either in vitro or by transplantation on (male) nude mice. Androgen receptor mRNA was clearly detectable in three androgen-dependent (sensitive) tumors and absent or low in three androgen-independent tumors. Growth of the LNCaP prostate tumor cell line can be stimulated both by androgens and by fetal calf serum. In the former situation androgen receptor mRNA expression is downregulated, whereas in the latter no effect on androgen receptor mRNA levels can be demonstrated. Sequence analysis showed that the androgen receptor gene from LNCaP cells contains a point mutation in the region encoding the steroid-binding domain, which confers an ACT coVon encoding a threonine residue to GCT, encoding alanine.     

v-rasH expression confers hormone-independent in vitro growth to LNCaP prostate carcinoma cells

The LNCaP human prostate cancer cell line is dependent on androgen for in vitro growth. To discover genes that may be responsible for progression of prostate cancer from hormone dependence to hormone independence, we transfected LNCaP cells with expression vectors that contained either the v-rasH or c-rasH gene under the control of the cadmium (Cd2+)-inducible human metallothionein-IIA promoter. Numerous derivative cell lines were isolated which manifested inducible expression of rasH p21 protein when the cells were treated with Cd2+. None of the cell lines transfected with c-rasH were found to have an altered growth phenotype. Several derivative cell lines expressing inducible v-rasH manifested hormone-independent growth in culture when treated with 10(-7) M Cd2+ . Cd2+ induction of v-rasH p21 was also shown to increase anchorage-independent colony formation of the v-rasH-expressing cell lines tested. Expression of a dominant mutated oncogene can change the hormone-dependent growth phenotype of prostate cancer cells.     

Somatostatin Derivative (smsDX) Targets Cellular Metabolism in Prostate Cancer Cells after Androgen Deprivation Therapy

Cancer cell metabolism responsive to androgen deprivation therapy (ADT) may be involved in the development and progression of prostate cancer and the ultimate failure of androgen-deprivation therapy. To investigate the metabolism regulation effects on androgen-independent growth of prostate cancer, an established LNCaP-s cell model that resembles the clinical scenario of castration-resistant prostate cancer (CRPC), was used in this current study. This cell line was cultured from androgen-sensitive LNCaP parental cells, in an androgen-reduced condition, resembling clinical androgen deprivation therapy. To assess the effects of smsDX on the invasiveness of prostate cancer cells we used wound healing assay and Matrigel™ invasion assay. We evaluated differentially expressed proteins of the parental LNCaP cells and LNCaP-s cells after ADT by means of two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF mass spectrometric analysis. The covered area in the wound and the number of cells invading through a Matrigel chamber were significantly smaller for cells treated with smsDX than they were for control cells treated with vehicle. 56 proteins were found differentially expressed in LNCaP-s cells compared to LNCaP cells, majority of them were down-regulated after ADT treatment. 104 proteins of LNCaP cells and 86 in LNCaP-s cells, separately, were found differentially expressed after treatment with smsDX, When we explored these protein functions within the website UniProtKB/Swiss-Prot, surprisingly, most of the proteins were found to be involved in the cellular metabolism and mitochondrial function regulation. LNCaP-s as potential metastatic androgen-independent cancer cells, its metabolism and mitochondrial functions could be altered by a new somatostatin derivative smsDX, the smsDX regulatory effects on metabolism in LNCaP-s deliver more therapeutic information with the treatment of CRPC.     

Requirement of androgen-dependent activation of protein kinase Czeta for androgen-dependent cell proliferation in LNCaP Cells and its roles in transition to androgen-independent cells

A cell line that we designed, AILNCaP, proliferated in androgen-depleted medium after emerging from long-term androgen-depleted cultures of an androgen-sensitive prostate cancer cell line, LNCaP. Using this cell line as a model of progression to androgen independence, we demonstrated that the activity of the mammalian target of rapamycin/p70 S6 kinase transduction pathway is down-regulated after androgen depletion in LNCaP, whereas its activation is related to transition of this cell line to androgen-independent proliferation. Kinase activity of protein kinase Czeta is regulated by androgen stimulation in LNCaP cells, whereas it is activated constitutively in AILNCaP cells under androgen-depleted conditions. Treatment with a protein kinase Czeta pseudosubstrate inhibitor reduced p70 S6 kinase activity and cell proliferation in both cell lines. We identified that both protein kinase Czeta and p70 S6 kinase were associated in LNCaP cells and this association was enhanced by the androgen stimulation. We examined the expression of phospho-protein kinase Czeta and phospho-p70 S6 kinase in hormone-naive prostate cancer specimens and found that the expression of both kinases was correlated with each other in those specimens. Significant correlation was observed between the expression of both kinases and Ki67 expression. Most of the prostate cancer cells that survived after prior hormonal treatment also expressed both kinases. This is the first report that shows the significance of this pathway for both androgen-dependent and -independent cell proliferation in prostate cancer. Our data suggest that protein kinase Czeta/mammalian target of rapamycin/S6 kinase pathway plays an important role for the transition of androgen-dependent to androgen-independent prostate cancer cells.