Down-regulation of homeobox gene GBX2 expression inhibits human prostate cancer clonogenic ability and tumorigenicity.

Previously, we have demonstrated that GBX genes, a homeobox-containing human family of DNA-binding transcription factors consisting of GBX1 and GBX2, are overexpressed in a panel of human prostatic cancer cell lines (ie., TSU-pr1, PC3, DU145, and LNCaP) compared to normal prostate. In the present studies, specific primer sets were designed for reverse transcription-PCR detection of the expression of GBX1 versus GBX2 in human prostate cancer. These studies demonstrated that the GBX2 gene, but not the GBX1 gene, is consistently overexpressed in this panel of human prostate cancer cell lines compared to normal human prostate. Using a quantitative-competitive PCR analysis, GBX2 mRNA was expressed as 3 x 10(3) copies/microg RNA in normal prostate tissue and 4 x 10(4) copies/microg RNA in the immortalized normal neonatal prostate epithelial cell line 267B-1, as compared to 6 x 10(5), 5 x 10(5), 3 x 10(5), and 1 x 10(5) copies/microg RNA in TSU-pr1, DU145, LNCaP, and PC3 prostate cancer cell lines, respectively. To examine the importance of GBX2 expression for prostate cancer malignancy, GBX2-overexpressing TSU-pr1 and PC3 human prostatic cancer cells were transfected with a eukaryotic expression vector containing an antisense GBX2 homeobox domain cDNA. Stable transfectant clones with 5-10-fold decreased levels of GBX2 mRNA expression were obtained. When tested in vitro, the clonogenic ability of the GBX2 antisense transfectants was reduced by approximately 50% in both cell lines. When implanted s.c. into nude mice, the tumorigenicity of the antisense GBX2 transfectants from both human prostatic cancer cell lines was inhibited by more than 70% compared to the parental cells. These results suggest that expression of GBX2 gene is required for malignant growth of human prostate cells.

CD44 is a metastasis suppressor gene for prostatic cancer located on human chromosome 11p13.

We have used microcell fusion-mediated chromosomal transfer to introduce normal human chromosomes into highly metastatic rodent prostatic cancer cells to map the location of a metastasis suppressor gene(s). Using this approach, several chromosomal regions have been identified that harbor such metastatic suppressor genes, including human chromosome 11 between p11.2-13 (T. Ichikawa et al., Cancer Res., 52: 3486-3490, 1992, 54: 2299-2302, 1994; N. Nihei et al., Genes Chromosomes & Cancer, 14: 112-119, 1995; C. W. Rinker-Schaeffer et al., Cancer Res., 54: 6249-6256, 1994). Using positional cloning, a metastatic suppressor gene, termed KAI1, was identified, which is located at human chromosome 11p11.2 (5). Overexpression of KAI1 results in metastasis suppression in certain highly metastatic Dunning R-3327 rat prostatic cancer sublines, such as AT6.1, without metastasis suppression in other highly metastatic sublines, such as AT3.1. This suggests that an additional metastasis suppressor gene is located within the human chromosome 11p11.2-13 region. The CD44 gene is located on human chromosome 11p13 and encodes an integral membrane glycoprotein that participates in specific cell-cell and cell-extracellular matrix interactions. Down-regulation of CD44 expression both at the mRNA and protein levels correlates with metastatic potential within the Dunning system of rat prostatic cancer sublines. Transfection-induced enhanced expression of the Mr 85,000 standard form of CD44 in the highly metastatic AT3.1 rat prostatic cells greatly suppresses their metastatic ability to the lungs without suppression of their in vivo growth rate or tumorigenicity. These results suggest that CD44 is a metastasis suppressor for prostatic cancer and that decreased expression of the standard form of CD44 is involved in the progression of prostatic cancer to a metastatic state.

Metastasis suppression by the standard CD44 isoform does not require the binding of prostate cancer cells to hyaluronate.

Previous studies from this laboratory have demonstrated that down-regulation of the standard CD44 isoform at the mRNA and protein level is associated with the acquisition of high metastatic ability within the Dunning R-3327 system of rat prostate cancers. Additional studies demonstrated that transfection-induced enhanced expression of the standard CD44 isoform suppresses the metastatic ability of the AT3.1 Dunning subline without suppressing tumorigenicity. The standard CD44 isoform is a major cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronate. In this study, an investigation was made to resolve whether the ability of the standard CD44 isoform to suppress metastasis of the AT3.1 prostate cancer cells critically requires enhanced hyaluronate binding. Highly metastatic Dunning AT3.1 rat prostate cancer cells were transfected with expression plasmids encoding either the wild-type or mutant standard CD44 isoform. The mutant standard CD44 isoform construct encoded a protein unable to bind to hyaluronate. Transfectants were isolated and characterized with regard to their level of standard CD44 isoform expression, hyaluronate binding, tumorigenicity, and metastatic ability. Expression of the wild-type standard CD44 isoform increased the hyaluronate binding of prostate cancer cells and suppressed their metastatic ability without suppressing their tumorigenicity. Expression of the mutant CD44 standard isoform did not increase hyaluronate binding; however, it equally suppressed the metastatic ability of the AT3.1 prostate cancer cells. These results demonstrate that the metastasis suppression by the standard CD44 isoform is independent of its ability to bind to hyaluronate.

Inhibition of constitutively activated Stat3 signaling pathway suppresses growth of prostate cancer cells.

Overexpression of interleukin 6, a downstream target of the GBX2 homeobox gene, has been linked to the progression of prostate cancer. The Janus kinase-signal transducers and activators of transcription signaling pathway transmits interleukin 6-mediated signals from cell surface receptors to the target genes in the nucleus and is critical in mediating cellular growth and differentiation. We demonstrate that cells derived from both rat and human prostate cancers have constitutively activated Stat3, with Stat3 activation being correlated with malignant potential. Blockade of activated Stat3 by ectopic expression of a dominant-negative Stat3 in human prostate cancer cells significantly suppresses their growth in vitro and their tumorigenicity in vivo. Furthermore, the Janus kinase inhibitor, tyrphostin AG490, inhibited the constitutive activation of Stat3 and suppressed the growth of human prostate cancer cells. These results indicate that activation of Stat3 signaling is essential in the progression of prostate cancer cells and suggest that targeting Stat3 signaling may yield a potential therapeutic intervention for prostate cancer.

Methylation of the CD44 metastasis suppressor gene in human prostate cancer.

Previous studies demonstrated that CD44 is a metastasis suppressor gene for prostate cancer and that the expression of CD44 both at mRNA and protein levels is down-regulated during prostate cancer progression, with down-regulation being correlated with higher tumor grade, aneuploidy, and distant metastasis. In this study, we evaluated DNA hypermethylation as a potential mechanism accompanying this decreased CD44 expression in human prostate cancer. Nucleotide sequence analysis revealed a CpG island in the CD44 transcriptional regulatory region. We found that cytosine methylation of CD44 promoter occurs in CD44-negative prostate cancer cell line (i.e., LNCaP) but not in prostate cancer cell lines (i.e., TSU, PC3, and DU145) expressing this gene. In addition, we examined methylation status of CD44 in 84 matched normal and cancer prostate specimens. Hypermethylation of the 5' CpG island of CD44 gene was observed in 31 of 40 primary prostate cancer specimens, 3 of 4 distant organ site metastases obtained at autopsy from men who died of prostate cancer, and 4 of the 40 matched normal tissues. These results demonstrated that methylation of the 5' CpG island of CD44 gene is closely associated with transcriptional inactivation, resulting in a decreased expression of CD44 in human prostate cancer.

Enhanced GBX2 expression stimulates growth of human prostate cancer cells via transcriptional up-regulation of the interleukin 6 gene.

Previous studies demonstrated that the GBX2 homeobox gene is consistently overexpressed in cultured human prostate cancer cell lines. In this study, the human GBX2 cDNA was cloned and a quantitative reverse transcription-PCR method used to demonstrate that GBX2 mRNA expression is enhanced in approximately 70% of human prostate cancer tissues compared with normal human prostate tissues. Purified recombinant GBX2 protein binds specifically to an ATTA motif within the promoter of the interleukin 6 (IL-6) gene. Using an antisense approach, down-regulation of the expression of GBX2 correlated with decreased expression of IL-6 and an inhibition of tumorigenicity of PC3 human prostate cancer cells. In addition, in vitro growth of the antisense clones was partially restored by exogenous addition of recombinant IL-6 protein to the culture media. These data demonstrated that enhanced GBX2 expression results in a stimulation of malignant growth of prostate cancer cells and that part of this stimulation involves up-regulation in the transcription of the IL-6 gene.

Targeting autophagy overcomes Enzalutamide resistance in castration-resistant prostate cancer cells and improves therapeutic response in a xenograft model.

Macro-autophagy is associated with drug resistance in various cancers and can function as an adaptive response to maintain cell survival under metabolic stresses, including androgen deprivation. Androgen deprivation or treatment with androgen receptor (AR) signaling inhibitor (ARSI), Enzalutamide (MDV-3100, ENZA) or bicalutamide induced autophagy in androgen-dependent and in castration-resistant CaP (castration-resistant prostate cancer (CRPC)) cell lines. The autophagic cascade triggered by AR blockage, correlated with the increased light chain 3-II/I ratio and ATG-5 expression. Autophagy was observed in a subpopulation of C4-2B cells that developed insensitivity to ENZA after sustained exposure in culture. Using flow cytometry and clonogenic assays, we showed that inhibiting autophagy with clomipramine (CMI), chloroquine or metformin increased apoptosis and significantly impaired cell viability. This autophagic process was mediated by AMP-dependent protein kinase (AMPK) activation and the suppression of mammalian target of rapamycin (mTOR) through Raptor phosphorylation (Serine 792). Furthermore, small interfering RNA targeting AMPK significantly inhibited autophagy and promoted cell death in CaP cells acutely or chronically exposed to ENZA or androgen deprivation, suggesting that autophagy is an important survival mechanism in CRPC. Lastly, in vivo studies with mice orthotopically implanted with ENZA-resistant cells demonstrated that the combination of ENZA and autophagy modulators, CMI or metformin significantly reduced tumor growth when compared with control groups (P<0.005). In conclusion, autophagy is as an important mechanism of resistance to ARSI in CRPC. Antiandrogen-induced autophagy is mediated through the activation of AMPK pathway and the suppression of mTOR pathway. Blocking autophagy pharmacologically or genetically significantly impairs prostate cancer cell survival in vitro and in vivo, implying the therapeutics potential of autophagy inhibitors in the antiandrogen-resistance setting.

Expression of homeobox gene-GBX2 in human prostatic cancer cells.

BACKGROUND: Homeobox genes encode transcription factors involved in the genetic control of normal development and differentiation, as well as in malignant transformation. To begin to assess the possible role of homeobox genes in prostatic cell carcinogenesis, we surveyed initially for expression of homeobox-containing genes in the TSU-PR1 cell line. METHODS: This was performed by RT-PCR using degenerate oligodeoxyribonucleotide primers to the homeobox-binding sequence to generate partial cDNAs which were cloned and sequenced. RESULTS: Using this method, expression of 14 members of homeobox-containing genes were detected in TSU-PR1 cells. All of these expressed genes correspond to previously identified homeobox genes located within the HOXA, B, C, and D clusters. We further examined the expression of these homeobox genes in different human prostatic cell lines by using whole cDNA slot blot and Northern blot analysis. One of the sequences corresponding to the human GBX2 homeobox gene is overexpressed in TSU-PR1, LNCaP, PC-3, and DU145 metastatic prostate cell lines relative to the normal prostate. CONCLUSIONS: Our results suggest that the homeobox gene GBX2 may participate in metastatic progression in prostatic cancer.

Identification of the rat homologue of KAI1 and its expression in Dunning rat prostate cancers.

BACKGROUND: We previously isolated the human KAI1 gene encoding a transmembrane protein which suppresses metastatic ability in Dunning R3327 AT6.1 rat prostate cancer cells when transfected into these cells. The AT6.1 subline is one of the more aggressive sublines among the Dunning R-3327 system of rat prostate cancers. This raises the issue of whether downregulation of KAI1 expression consistently occurs during the acquisition of high metastatic ability by members of the Dunning system of rat prostate cancers. METHODS: To investigate this possibility, the rat homologue of the KAI1gene was identified, using a combination of cDNA library screening and 5'-RACE and DNA sequencing. Based on this information, a rat-specific cDNA probe was developed and used for Northern blot analysis of KAI1 expression in normal rat tissues and a series of sublines of Dunning R3327 cells that vary widely in their metastatic abilities. RESULTS: The rat KAI1 gene encoded a protein of 266 amino acids which has 77% identity to the human KAI1 protein. In normal tissues, KAI1 is expressed predominantly as a 2.0-kb-sized transcript. Several tissues (e.g., skeletal muscle and prostate) also express a minor 1.8-kb-sized RNA. Northern blot analysis of a series of Dunning sublines demonstrated that all sublines expressed both the 2.0- and 1.8-kb KAI1 RNA transcripts. However, quantitative levels of the 2.0- vs. 1.8-kb KAI1 RNA were variable among sublines. Downregulation of expression of the 2.0-kb KAI1 transcript was statistically correlated with the acquisition of high metastatic ability within this system of prostate cancer sublines. In contrast, the 1.8-kb transcript was upregulated in all of the more aggressive sublines, but this enhanced expression was not specifically correlated with metastatic ability. CONCLUSIONS: These studies demonstrated that downregulation of the 2.0-kb KAI1 mRNA is associated with the acquisition of high metastatic ability by prostate cancer cells.

Interleukin-6 induces prostate cancer cell growth accompanied by activation of stat3 signaling pathway .

BACKGROUND: Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates growth and differentiation of various types of malignant tumors, including prostate carcinomas. The levels of IL-6 are elevated in sera of patients with metastatic prostate cancer. In this study, we evaluate the role of IL-6 in the growth regulation of prostate cancer cells. METHODS: Expression of IL-6 and its receptors in human prostate cancer cells was measured by ELISA and RT-PCR. The effects of IL-6 on cell growth were evaluated by ectopically expressing IL-6 cDNA into IL-6-negative LNCaP human prostate cancer cells. Stat3 DNA binding activities were analyzed by electromobility shift assay and supershift assay. RESULTS: Expression of IL-6 was detected in the androgen-insensitive prostate cancer cell lines (i.e. , TSU, PC3, and DU145), but not in the androgen-sensitive LNCaP cell line. IL-6 receptors, including both IL-6-specific receptor alpha chain and gp130 signal transducer, are expressed in all human prostate cancer cell lines (i.e., LNCaP, TSU, PC3, and DU145). Overexpression of IL-6 by ectopically expressing IL-6 into IL-6-negative LNCaP human prostate cancer cells significantly increased clonogenic ability and cell proliferation in vitro compared to the IL-6-negative parental LNCaP cells and the antisense controls. This growth stimulation by IL-6 was accompanied by activation of the Stat3 signaling transduction pathway. CONCLUSIONS: IL-6 is an autocrine growth factor for LNCaP human prostate cancer cells; the effects of IL-6 on prostate cancer cell growth are mediated through the Janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling pathway.

Suppression of the tumorigenicity of prostatic cancer cells by gene(s) located on human chromosome 19p13.1-13.2.

BACKGROUND: In previous reports, we used microcell fusion-mediated chromosomal transfer to introduce normal human chromosomes into highly metastatic rat prostatic cancer cells to map the location of tumor and metastasis suppressor genes. The gene for prostate-specific antigen as well as several classes of genes, including cell adhesion molecules, previously demonstrated to be altered during prostate cancer progression, were mapped to human chromosome 19. METHODS: A normal human chromosome 19 was introduced into Dunning-R3327 AT6.1 rat and TSU-prl human prostatic cancer cells by microcell-mediated chromosome transfer to test the suppressive effects of this chromosome on prostate cancer. Five independent hybrid clones from Dunning-R3327 AT6.1 rat prostatic cancer cells and four independent hybrid clones from TSU-pr1 human prostatic cancer cells were isolated, karyotyped, allelotyped, and analyzed for in vitro and in vivo growth characteristics. RESULTS: Introduction of human chromosome 19 into both the rat and human prostatic cancer cells resulted in alteration of cell morphology in vitro and suppression of tumorigenicity in vivo in athymic nude mice. Highly polymorphic SSR2 markers mapped to human chromosome 19 were used to determine the portions of human chromosome 19 retained in the hybrids. These analyses identified a region localized on human chromosome 19p13.1-13.2 that is responsible for the tumor suppression of both rat and human prostatic cancer cells. The expression of several genes previously mapped to this human chromosome 19p13.1-13.2 region (i.e., ICAM-1, Notch3, and Stau) were analyzed to evaluate if they could be candidate suppressor genes for prostate cancer cell growth in vivo, but no expression patterns consistent with those predicted for a suppressor gene were observed. CONCLUSIONS: Human chromosome 19p13.1-13.2 contains potential tumor suppressor gene(s) for prostate cancer.