Imaging, procedural and clinical variables associated with tumor yield on bone biopsy in metastatic castration-resistant prostate cancer.

BACKGROUND: Understanding the mechanisms driving disease progression is fundamental to identifying new therapeutic targets for the treatment of men with metastatic castration-resistant prostate cancer (mCRPC). Owing to the prevalence of bone metastases in mCRPC, obtaining sufficient tumor tissue for analysis has historically been a challenge. In this exploratory analysis, we evaluated imaging, procedural and clinical variables associated with tumor yield on image-guided bone biopsy in men with mCRPC. METHODS: Clinical data were collected prospectively from men with mCRPC enrolled on a phase II trial with serial metastasis biopsies performed according to standard clinical protocol. Imaging was retrospectively reviewed. We evaluated the percent positive biopsy cores (PPC), calculated as the number of positive cores divided by the total number of cores collected per biopsy. RESULTS: Twenty-nine men had 39 bone biopsies. Seventy-seven percent of bone biopsies had at least one positive biopsy core. We determined that lesion size and distance from the skin to the lesion edge correlated with tumor yield on biopsy (median PPC 75% versus 42% for lesions >8.8 cm(3) versus ⩽ 8.8 cm(3), respectively, P=0.05; median PPC 33% versus 71% for distance ⩾ 6.1 versus <6.1 cm, respectively, P = 0.02). There was a trend towards increased tumor yield in patients with increased uptake on radionuclide bone scan, higher calcium levels and shorter duration of osteoclast-targeting therapy, although this was not statistically significant. Ten men had 14 soft tissue biopsies. All soft tissue biopsies had at least one positive biopsy core. CONCLUSIONS: This exploratory analysis suggests that there are imaging, procedural and clinical variables that have an impact on image-guided bone biopsy yield. In order to maximize harvest of prostate cancer tissue, we have incorporated a prospective analysis of the metrics described here as part of a multi-institutional project aiming to use the molecular characterization of mCRPC tumors to direct individual therapy.

Identification of multiple cis-acting elements mediating the induction of prostaglandin G/H synthase-2 by phorbol ester in murine osteoblastic cells.

The tumor promoter phorbol 13-myristate 12-acetate (PMA), the best characterized protein kinase C agonist, frequently regulates gene expression via activation of Fos/Jun (AP-1) complexes. PMA rapidly and transiently induces prostaglandin G/H synthase-2 (PGHS-2) expression in murine osteoblastic MC3T3-E1 cells, but no functional AP-1 binding motifs in the 5'-flanking region have been identified. In MC3T3-E1 cells transfected with -371/+70 bp of the PGHS-2 gene fused to a luciferase reporter gene (Pluc), PMA stimulates luciferase activity up to eightfold. Computer analysis of the sequence of the PGHS-2 promoter region identified three potential AP-1 elements in the -371/+70 bp region, and deletion analysis suggested that the sequence 5'-aGAGTCA-3' at -69/-63 bp was most likely to mediate stimulation by PMA. Mutation of the putative AP-1 sequence reduces the ability of PMA to stimulate Pluc activity by 65%. On electrophoretic mobility shift analysis (EMSA), PMA induces binding to a PGHS-2 probe spanning this sequence, binding is blocked by an unlabeled AP-1 canonical sequence, and antibodies specific for c-Jun and c-Fos inhibit binding. Mutation of this AP-1 site also causes a small (22%) but significant reduction in the serum stimulation of Pluc activity in transiently transfected MC3T3-E1 cells. On EMSA, serum induces binding to a PGHS-2 probe spanning the AP-1 site, binding is blocked by an unlabeled AP-1 canonical sequence, and antibodies specific for c-Jun and c-Fos inhibit binding. Joint mutation of this AP-1 site and the nearby CRE site at -56/-52 bp, previously shown to mediate serum, v-src and PDGF induction of PGHS-2 in NIH-3T3 cells, blocks both PMA and serum induction of Pluc activity in MC3T3-E1 cells. Hence, the AP-1 and CRE binding sites are jointly but differentially involved in both the PMA and serum stimulation of PGHS-2 promoter activity.

Transforming growth factor-beta1 regulation of prostaglandin G/H synthase-2 expression in osteoblastic MC3T3-E1 cells.

Transforming growth factor-beta (TGFbeta) plays an important role in bone development and remodeling. TGFbeta stimulates PGE2 production, enhances interleukin-1-stimulated PGE2 production, and can stimulate PG-mediated bone resorption. We found that TGFbeta induced prostaglandin G/H synthase (PGHS-2) messenger RNA (mRNA) and PGE2 production in neonatal mouse calvarial cultures and in primary cells derived from these calvariae. We used MC3T3-E1 cells, an immortalized osteoblastic cell line derived from mouse calvariae, to examine the mechanism of PGHS-2 induction. PGHS-2 mRNA was rapidly induced by TGFbeta (10 ng/ml) in MC3T3-E1 cells; mRNA levels peaked at 4-8 h and were still elevated at 24 h. Induction of PGHS-2 protein and PGE2 production correlated with PGHS-2 mRNA levels. In contrast, TGFbeta had much less effect on PGHS-1 mRNA levels. Unlike the response to other agonists, PGHS-2 mRNA induction by TGFbeta was not enhanced by cycloheximide pretreatment, suggesting a requirement for new protein synthesis. To study transcriptional regulation, cells were stably transfected with a PGHS-2 promoter-luciferase reporter construct containing 371 bp of the 5'-flanking region and 70 bp of untranslated DNA from the PGHS-2 gene. TGFbeta-stimulated luciferase activity paralleled PGHS-2 mRNA induction. Stimulation of luciferase activity and PGHS-2 mRNA levels by other agonists, including interleukin-1, TGF alpha, forskolin, and phorbol 13-myristate 12-acetate, were enhanced by TGFbeta. A 90% drop in luciferase activity occurred with deletion of the region from -371 to -213 bp of the PGHS-2 promoter. The PG response to TGFbeta in MC3T3-E1 cells appears to be mediated primarily by transcriptional regulation of PGHS-2 expression through one or more cis-acting elements located between -371 and -213 bp in the 5'-flanking region of the PGHS-2 gene.

Interleukin-4 inhibits prostaglandin G/H synthase-2 and cytosolic phospholipase A2 induction in neonatal mouse parietal bone cultures.

We have shown previously that prostaglandin (PG) production in 7-day-old neonatal mouse calvarial cultures is regulated largely by changes in prostaglandin G/H synthase-2 (PGHS-2) expression and to a lesser extent by changes in arachidonic acid (AA) release. In this study, we examined the effects of interleukin-4 (IL-4), and its interactions with other cytokines and with parathyroid hormone (PTH), on mRNA levels of PGHS-2, PGHS-1, and cytosolic phospholipase A2 (cPLA2) and on medium protaglandin E2 (PGE2) levels in calvarial cultures. IL-1 and tumor necrosis factor-alpha (TNF-alpha), both at 1-100 ng/ml, and PTH at 0.1-10 nM increased PGHS-2 and cPLA2 mRNA and medium PGE2 levels dose-dependently after 4 h of treatment. IL-6 and IL-11 at 1-100 ng/ml did not affect mRNA or PGE2 levels. IL-4 at 1-100 ng/ml decreased PGHS-2 and cPLA2 mRNA and PGE2 levels in control as well as IL-1, TNF-alpha, and PTH-stimulated cultures. The inhibition of PGHS-2 and cPLA2 mRNA expression by IL-4 (10 ng/ml) was present at 1 h, reached a maximum at 4 h, and persisted for 24 h. The effects were maintained in the presence of cycloheximide. IL-4 also decreased PGHS-2 protein levels in control and IL-1-stimulated cultures. PGHS-1 mRNA levels were not stimulated by any of the factors studied nor inhibited by IL-4. IL-4 partially inhibited control and PTH-stimulated 45Ca release from prelabeled mouse calvariae at 4 days. However, neither the inhibition of resorption by IL-4 nor the stimulation by IL-1 and PTH were altered by indomethacin (1 microM). We conclude that (1) IL-1, TNF-alpha, and PTH, but not IL-6 nor IL-11, can increase the expression of PGHS-2, cPLA2, and PGE2 production in cultured mouse calvariae; (2) IL-4 inhibits PGE2 production in both control and stimulated calvarial cultures by inhibiting PGHS-2 and cPLA2; and (3) IL-4 has an inhibitory effect on bone resorption which is independent of PG production.

Autoregulation of inducible prostaglandin G/H synthase in osteoblastic cells by prostaglandins.

Prostaglandins (PGs) have been postulated to amplify their own production by stimulating cyclic adenosine monophosphate activity, which in turn stimulates PG production. We examined regulation of messenger RNA levels for the inducible and constitutive prostaglandin G/H synthases, PGHS-2 and PGHS-1, in murine osteoblastic MC3T3-E1 cells, which express both PGHS-1 and PGHS-2, and in rat osteoblastic Py1a cells, which express only PGHS-2. Prostaglandins E2, F2 alpha, and D2 induced PGHS-2 mRNA in both cell lines under serum-free conditions and stimulated small increases in PGHS-1 mRNA levels in MC3T3-E1 cells. PGE2 (1 microM) increased the transcription rate of PGHS-2 mRNA 9-fold at 2 h in serum-free cells and also induced PGHS-2 protein. In the presence of arachidonic acid or serum, PGs also increased medium PGE2. Both forskolin, a protein kinase A activator, and phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, have previously been shown to induce PGHS-2 mRNA in MC3T3-E1 cells, but in the present study only PMA induced PGHS-2 expression in Py1a cells. The induction of PGHS-2 mRNA in Py1a cells by PGs was inhibited by chelerythrine, a PKC inhibitor, and blocked by 24 h of pretreatment with PMA. The 2 h serum stimulation of PGHS-2 mRNA in MC3T3-E1 cells was inhibited 40-50% by three structurally unrelated nonsteroidal anti-inflammatory drugs (NSAIDs), suggesting that endogenous PGs also amplify PG production through induction of PGHS-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the two prostaglandin G/H synthases by parathyroid hormone, interleukin-1, cortisol, and prostaglandin E2 in cultured neonatal mouse calvariae.

A second prostaglandin G/H synthase (PGHS-2), encoded by a gene separate from that for the original PGHS (PGHS-1), has recently been identified. We have shown that PGHS-2 is expressed in cultured mouse calvariae and have compared regulation of PGHS-2 and PGHS-1 messenger RNA (mRNA) levels. PGHS-2 mRNA was not detectable in freshly isolated bones, but was induced during culture and further stimulated by interleukin-1 (IL-1) and PTH. Both factors also increased PGHS-2 protein levels. Changes in medium prostaglandin E2 (PGE2) production correlated with increases in PGHS-2 mRNA levels. However, with IL-1, PGE2 production was increased more than PGHS-2 mRNA levels (treated/control ratio, 3.4 and 1.5, respectively), whereas with PTH there was a closer correspondence (2.0 and 2.1). Cortisol reduced PTH-stimulated PGE2 production (treated/control ratio decreased from 3.1 to 0.2) more than PGHS-2 mRNA levels (2.8 to 0.8). In the presence of exogenous arachidonic acid, changes in PGHS-2 mRNA levels with IL-1, PTH, and cortisol correlated closely with changes in PGE2 production. PGE2 itself increased PGHS-2 mRNA, and nonsteroidal antiinflammatory drugs decreased PGHS-2 mRNA levels by 80%. In contrast, PGHS-1 mRNA was expressed constitutively and was not affected by IL-1, PTH, or cortisol when measured by competitive reverse transcriptase-polymerase chain reaction. We conclude that regulation of PGE2 production is predominantly through PGHS-2, rather than PGHS-1; that IL-1 and cortisol may also regulate arachidonic acid release; and that PGE2 may amplify its own production through stimulation of PGHS-2.

Differential regulation of inducible and constitutive prostaglandin endoperoxide synthase in osteoblastic MC3T3-E1 cells.

Regulation of mRNA levels for the constitutive and inducible prostaglandin endoperoxide synthases, PGHS-1 and PGHS-2, was examined in murine osteoblastic MC3T3-E1 cells. Serum induction of PGHS-2 mRNA levels was rapid, transient, increased by cycloheximide, and inhibited 72% by cortisol. The cortisol inhibition was blocked by cycloheximide. Serum stimulation of PGHS-1 mRNA was slower, decreased by cycloheximide, and inhibited 28% by cortisol. Increased prostaglandin E2 (PGE2) production and induction of PGHS-2 immunoreactive protein paralleled changes in PGHS-2 mRNA. PGHS-2 mRNA was induced at 2 h in serum-free cells by transforming growth factor-beta (TGF-beta), phorbol 12-myristate 13-acetate, and, to a lesser extent, by forskolin. The combination of phorbol 12-myristate 13-acetate and forskolin was synergistic. TGF-beta induction was prolonged compared with serum, inhibited 67% by cortisol, and the inhibition was not blocked by cycloheximide. TGF-alpha had little effect on PGHS-2 mRNA at 2 h, but the combination of TGF-beta and TGF-alpha was synergistic for PGHS-1 and PGHS-2. PGE2 itself induced PGHS-2 mRNA, and inhibition of PGE2 production decreased the serum induction by 55%, suggesting an important role for autoamplification. The rapidity and amplitude of changes in PGHS-2 suggest that it may be involved in bone responses to acute stresses, such as mechanical strain, inflammation, and injury.