Circulating microRNAs as potential new biomarkers for prostate cancer.

Since they were first described in the 1990s, circulating microRNAs (miRNAs) have provided an active and rapidly evolving area of current research that has the potential to transform cancer diagnostics and therapeutics. In particular, miRNAs could provide potential new biomarkers for prostate cancer, the most common cause of cancer in UK men. Current diagnostic tests for prostate cancer have low specificity and poor sensitivity. Further, although many prostate cancers are so slow growing as not to pose a major risk to health, there is currently no test to distinguish between these and cancers that will become aggressive and life threatening. Circulating miRNAs are highly stable and are both detectable and quantifiable in a range of accessible bio fluids, thus have the potential to be useful diagnostic, prognostic and predictive biomarkers. This review aims to summarise the current understanding of circulating miRNAs in prostate cancer patients and their potential role as biomarkers.

Endo180 modulation by bisphosphonates and diagnostic accuracy in metastatic breast cancer.

BACKGROUND: Endo180 (CD280; MRC2; uPARAP)-dependent collagen remodelling is dysregulated in primary tumours and bone metastasis. Here, we confirm the release and diagnostic accuracy of soluble Endo180 for diagnosing metastasis in breast cancer (BCa). METHODS: Endo180 was quantified in BCa cell conditioned medium and plasma from BCa patients stratified according to disease status and bisphosphonate treatment (n=88). All P-values are from two-sided tests. RESULTS: Endo180 is released by ectodomain shedding from the surface of MCF-7 and MDA-MB-231 BCa cell lines. Plasma Endo180 was significantly higher in recurrent/metastatic (1.71±0.87; n=59) vs early/localised (0.92±0.37; n=29) BCa (P<0.0001). True/false-positive rates for metastasis classification were: 85%/50% for the reference standard, CA 15-3 antigen (28 U ml(-1)); ≤97%/≥36% for Endo180; and ≤97%/≥32% for CA 15-3 antigen+Endo180. Bisphosphonate treatment was associated with reduced Endo180 levels in BCa patients with bone metastasis (P=0.011; n=42). True/false-positive rates in bisphosphonate-naive patients (n=57) were: 68%/45% for CA 15-3 antigen; ≤95%/≥20% for Endo180; and ≤92%/≥21% for CA 15-3 antigen+Endo180. CONCLUSION: Endo180 is a potential marker modulated by bisphosphonates in metastatic BCa.

Circulating sphingosine-1-phosphate and erythrocyte sphingosine kinase-1 activity as novel biomarkers for early prostate cancer detection.

BACKGROUND: Current markers available for screening normal populations and for monitoring prostate cancer (PCa) treatment lack sensitivity and selectivity. Sphingosine-1-phosphate (S1P) is a circulating lipid second messenger involved in cell growth and migration, the immune response, angiogenesis, and malignant transformation. METHODS: Eighty-eight patients with localised, locally advanced, or metastatic PCa were recruited into this prospective single-centre study. Plasma S1P levels were measured and compared with age-matched controls with benign prostate hyperplasia (BPH) (n=110) or with young healthy males with the very small chance of having PCa foci (n=20). RESULTS: Levels of circulating S1P were significantly higher in healthy subjects (10.36 ± 0.69 pmol per mg protein, P<0.0001) and patients with BPH (9.39 ± 0.75, P=0.0013) than in patients with PCa (6.89 ± 0.58, ANOVA, P=0.0019). Circulating S1P levels were an early marker of PCa progression to hormonal unresponsiveness and correlated with prostate-specific antigen (PSA) levels and lymph node metastasis. During the course of the study, nine patients have died of PCa. Importantly, their circulating S1P levels were significantly lower (5.11 ± 0.75) than in the surviving patients (7.02 ± 0.22, n=79, P=0.0439). Our data suggest that the decrease in circulating S1P during PCa progression may stem from a highly significant downregulation of erythrocyte sphingosine kinase-1 (SphK1) activity (2.14 ± 0.17 pmol per mg protein per minute in PCa patients vs 4.7 ± 0.42 in healthy individuals, P<0.0001), which may be a potential mechanism of cancer-induced anaemia. CONCLUSION: This current study has provided a potential mechanism for cancer-related anaemia and the first evidence that plasma S1P and erythrocyte SphK1 activity are the potential markers for the diagnosis, monitoring, and predicating for PCa mortality.

Dhrs3a regulates retinoic acid biosynthesis through a feedback inhibition mechanism.

Retinoic acid (RA) is an important developmental signaling molecule responsible for the patterning of multiple vertebrate tissues. RA is also a potent teratogen, causing multi-organ birth defects in humans. Endogenous RA levels must therefore be tightly controlled in the developing embryo. We used a microarray approach to identify genes that function as negative feedback regulators of retinoic acid signaling. We screened for genes expressed in early somite-stage embryos that respond oppositely to treatment with RA versus RA antagonists and validated them by RNA in situ hybridization. Focusing on genes known to be involved in RA metabolism, we determined that dhrs3a, which encodes a member of the short-chain dehydrogenase/reductase protein family, is both RA dependent and strongly RA inducible. Dhrs3a is known to catalyze the reduction of the RA precursor all-trans retinaldehyde to vitamin A; however, a developmental function has not been demonstrated. Using morpholino knockdown and mRNA over-expression, we demonstrate that Dhrs3a is required to limit RA levels in the embryo, primarily within the central nervous system. Dhrs3a is thus an RA-induced feedback inhibitor of RA biosynthesis. We conclude that retinaldehyde availability is an important level at which RA biosynthesis is regulated in vertebrate embryos.

Inflammatory cytokines and aromatase inhibitor-associated musculoskeletal syndrome: a case-control study.

BACKGROUND: The aromatase inhibitor (AI)-associated musculoskeletal syndrome (AIMSS) occurs in approximately 50% of AI-treated patients. Inflammatory mediators are associated with oestrogen signalling and may change with oestrogen depletion. We hypothesised that AIMSS may be associated with changes in circulating inflammatory markers. METHODS: Patients with breast cancer were enrolled in a trial of adjuvant AI therapy. Changes in pain and function during therapy were assessed prospectively. We selected 30 cases with AIMSS and 22 controls without AIMSS, matched for demographics and prior therapy. Serum samples collected at baseline and during treatment were assayed for multiple inflammatory cytokines and lipid mediators using multiplex assays. RESULTS: Before AI therapy, mean serum concentrations of 6 of 36 assayed factors were statistically significantly lower in cases than controls (all P<0.003). No statistically significant changes during AI therapy relative to pre-treatment were observed between cases and controls for any of the inflammatory markers tested. CONCLUSION: AIMSS is probably not associated with a systemic inflammatory response. Pre-treatment cytokine levels may predict for development of AIMSS.

Zebrafish wnt8 encodes two wnt8 proteins on a bicistronic transcript and is required for mesoderm and neurectoderm patterning.

In vertebrates, wnt8 has been implicated in the early patterning of the mesoderm. To determine directly the embryonic requirements for wnt8, we generated a chromosomal deficiency in zebrafish that removes the bicistronic wnt8 locus. We report that homozygous mutants exhibit pronounced defects in dorso-ventral mesoderm patterning and in the antero-posterior neural pattern. Despite differences in their signaling activities, either coding region of the bicistronic RNA can rescue the deficiency phenotype. Specific interference of wnt8 translation by morpholino antisense oligomers phenocopies the deficiency, and interference with wnt8 translation in ntl and spt mutants produces embryos lacking trunk and tail. These data demonstrate that the zebrafish wnt8 locus is required during gastrulation to pattern both the mesoderm and the neural ectoderm properly.

Secreted Frizzled-related protein-1 is a negative regulator of androgen receptor activity in prostate cancer.

Secreted Frizzled-related protein-1 (sFRP1) associates with Wnt proteins and its loss can lead to activation of Wnt/beta-catenin signalling. It is frequently downregulated in cancer, including prostate cancer, but its function in prostate cancer is unclear because it can increase proliferation of prostate epithelial cells. We investigated the function of sFRP1 in androgen-dependent prostate cancer and found that sFRP1 inhibited androgen receptor (AR) transcriptional activity. In addition, sFRP1 inhibited the proliferation of androgen-dependent LNCaP cells but not of an androgen-independent subline LNCaP-r, suggesting a role in androgen-dependent growth. The inhibition of AR by sFRP1 was unaffected by co-expression of Wnt3a, stabilised beta-catenin or beta-catenin shRNA, suggesting it does not involve Wnt/beta-catenin signalling. Wnt5a also inhibited AR and expression of Wnt5a and sFRP1 together did not further inhibit AR, suggesting that Wnt5a and sFRP1 activate the same signal(s) to inhibit AR. However, sFRP1 inhibition of AR was unaffected by inhibitors of kinases involved in Wnt/Ca(2+) and Wnt/planar cell polarity non-canonical Wnt signalling. Interestingly, the cysteine-rich domain of sFRP1 interacted with Frizzled receptors expressed in prostate cancer cells, suggesting that sFRP1/Frizzled complexes activate a signal that leads to repression of AR. Taken together, these observations highlight the function of beta-catenin-independent Wnt signalling in the control of AR activity and provide one explanation for sFRP1 downregulation in prostate cancer.

Prohibitin, a protein downregulated by androgens, represses androgen receptor activity.

Prohibitin (PHB) is a cell cycle regulatory protein, known to repress E2F1-mediated gene activation via recruitment of transcriptional regulatory factors such as retinoblastoma and histone deacetylase 1 (HDAC1). We previously identified PHB as a target protein of androgen signaling in prostate cancer cells and showed that downregulation of PHB is required for androgen-induced cell cycle entry in these cells. We now present evidence that PHB, which has 54% homology at the protein level to the oestrogen receptor corepressor REA (repressor of oestrogen receptor activity), can repress androgen receptor (AR)-mediated transcription and androgen-dependent cell growth. Depletion of endogenous PHB resulted in an increase in expression of the androgen-regulated prostate-specific antigen gene. The repression appears to be specific to androgen and closely related receptors, as it is also evident for the glucocorticoid and progesterone, but not oestrogen, receptors. In spite of interaction of PHB with HDAC1, HDAC activity is not required for this repression. Although AR and PHB could be co-immunoprecipitated, no direct interaction was detectable, suggesting that PHB forms part of a repressive complex with the AR. Competition with the co-activator SRC1 further suggests that formation of a complex with AR, PHB and other cofactors is the mechanism by which repression is achieved. It appears then that repression of AR activity is one mechanism by which PHB inhibits androgen-dependent growth of prostate cells. Further, this study implies that the AR itself could, by mediating downregulation of a corepressor, be involved in the progression of prostate tumours to the hormone refractory stage.

Regulation of prostate cell growth and morphogenesis by Dickkopf-3.

Wnt signalling plays a critical role in the development of cancer. Recent studies indicate that Wnt signalling is negatively regulated by secreted Wnt antagonists such as secreted frizzled related proteins (sFRPs) and Dickkopfs (Dkks). We compared Dkk family expression levels in normal prostate and prostate cancer cells and found a reduction in Dkk-3 expression in cancer cells. Ectopic expression of Dkk-3 inhibited colony formation in LNCaP and PC3 prostate cancer cell lines and inducible expression of Dkk-3 reduced LNCaP cell proliferation. Moreover, small interfering RNA-mediated downregulation of Dkk-3 enhanced cell cycle progression in untransformed RWPE-1 prostate epithelial cells. Immunohistochemical analysis revealed that Dkk-3 is expressed in a subset of normal prostate gland acini and that Dkk-3 expression is reduced in prostate tumours, particularly those with a high Gleason grade, suggesting a role for Dkk-3 in postmitotic differentiation. Consistent with this, depletion of Dkk-3 disrupted acinar morphogenesis of RWPE-1 cells in a three-dimensional cell culture model. Our results are consistent with the loss of Dkk-3 expression resulting in impairment of glandular structure and uncontrolled prostate epithelial cell (PrEC) proliferation, both of which are crucial for prostate cancer progression.

Levels of matrix metalloproteases in bladder cancer correlate with tumor grade and invasion.

We have used quantitative zymography to measure levels of the type IV collagenases matrix metalloproteinase (MMP)-9 and MMP-2 in 42 biopsies of transitional cell carcinoma and in 7 biopsies of normal bladder. Mean levels of MMP-9 were significantly higher in tumor compared with normal samples (P = 0.08). Levels of MMP-9 and active MMP-2 increased with tumor grade (test for trend, P = 0.002 and P = 0.05, respectively). Levels of MMP-9 and activated MMP-2 were also higher in invasive tumors than in superficial ones (P = 0.001 and P = 0.008, respectively). In situ hybridization studies showed that the mRNAs for both MMP-2 and MMP-9 were located chiefly in the stroma rather than epithelial tumor cells and were concentrated at the interface between the two tissues.

Introduction: Basic science aspects of prostate cancer

No abstract Copyright 1997Academic Press Limited

Short-term therapy for acute myelogenous leukemia.

Since 1978, 187 patients (age range, 15 to 59, median 44 years) have received short-term chemotherapy as part of three sequential open studies (B-IX, X, Xb) or a randomized clinical trial (B-XI). An intended six cycles of Adriamycin (ADR) (doxorubicin; Adria Laboratories, Columbus, OH), cytarabine (ara-C), and thioguanine (TG) were administered with as short an intercycle time as possible. No further therapy was administered. Complete remission (CR) was achieved in 118 of 187 patients (63%). On univariate and multivariate analyses achievement of CR correlated adversely with a low serum albumin at presentation and an antecedent marrow disorder. Forty-five patients continue in first remission between 15 months and 8 1/2 years, no relapses being seen after 3 1/2 years (median follow-up, 3 1/2 years). The median duration of remission was 1 year. M3 morphology, a blast count less than 100 x 10(9)/L, and absence of hepatosplenomegaly correlated favorably with remission duration. There was no difference in duration of remission between patients receiving 3, 4, 5, or 6 cycles. The best results overall were achieved in patients under the age of 40, with 43% projected to remain free of disease at 5 years. Fifty patients remain alive between 17 months and 9 years, the predicted actuarial survival being 25% at 5 years.

Prostatic complications of testosterone replacement therapy.

The prostatic complications of testosterone replacement therapy have received little clinical attention. We describe three hypogonadal men who had prostatic disease (adenocarcinoma in two) detected in relation to such therapy. Literature review suggests that surveillance for early prostate cancer is appropriate during replacement therapy in men over the age of 50 years. We discuss the selective use of digital rectal examination, transrectal sonography, directed prostate biopsy, and prostate-specific antigen determinations before therapy and in subsequent follow-up in this age group of men during androgen replacement.

Systemic lupus erythematosus (SLE) induced by quinidine.

Only two definite cases of quinidine-induced systemic lupus erythematosus (SLE) are reported in the English-language literature. We have treated five patients who had an SLE-like syndrome while receiving quinidine. Symptoms, signs, and abnormal laboratory values improved after quinidine therapy was discontinued and prednisone therapy was started. The disease did not return after steroids were withdrawn. These cases indicate that quinidine can indeed cause an SLE-like syndrome.

Effects of chemotherapy on fertility.

Over recent years radical improvements in the treatment of malignant disease have resulted in the cure of patients in or approaching reproductive age, and with that cure, the effects on gonadal function have become apparent. Gonadal failure is particularly important in Hodgkin's disease, germ cell tumours, choriocarcinoma, and leukaemia, diseases of young people which are largely curable. Premature ovarian and testicular failure are easily missed by doctors concerned primarily with the erradication or arrest of a life-threatening disease, but for the patient the hormonal, psychological and social sequelae of treatment may be devastating. In this article we review the effects of chemotherapy on gonadal function and discuss the management of gonadal failure.

Mechanisms of androgen receptor signalling via steroid receptor coactivator-1 in prostate.

The androgen receptor (AR) is a member of the nuclear receptor superfamily. These ligand-activated transcription factors usually contain two activation functions, a ligand-independent activation function 1(AF1) in the divergent N-terminal domain and a ligand-dependent AF2 in the more conserved C-terminal ligand-binding domain. To promote transcription from target promoters, DNA-bound nuclear receptors recruit coactivator proteins that promote transcription by modifying histones within nucleosomes, resulting in altered topology of chromatin to allow access of the basal transcriptional machinery, or stabilising the pre-initiation complex. It is well known that most coactivators interact with AF2 of many nuclear receptors via conserved, helical LxxLL motifs (where L is leucine and x is any amino acid). The AF2 of the AR is very weak, but we were able to demonstrate that its intrinsic ligand-dependent activity is potentiated by steroid receptor coactivator-1 (SRC1) and that this region interacts with coactivators via LxxLL motifs. However, a mutant SRC1 coactivator with no functional LxxLL motifs was still able to potentiate AR activity. We found that SRC1 can also be recruited to (and increase activity of) AF1 of the AR via a conserved, glutamine-rich region. Point mutations within this region abolish SRC1 interaction with AF1 and also abolish or severely impair its ability to potentiate AR activity on all promoters tested. Thus the AR interacts with SRC1 via two different regions and the AF1 interaction is functionally the more important, although the contribution of the two interactions varies in a promoter-dependent fashion. SRC1 then potentiates receptor activity via recruitment of CBP/p300, a histone acetyltranferase. This is important in the context of prostate cancer as SRC1 and other coactivators including CBP are coexpressed with AR in the luminal epithelial cells of the prostate, where over 90% of prostate tumours arise. There is a need for effective second-line prostate cancer therapy aimed at blocking the AR pathway when anti-androgen therapy has failed. Since there is growing evidence that nuclear receptor cofactors may be implicated in the progression of hormone-dependent tumours to hormone-independent states, novel targets could include the interaction of AR with coactivator proteins. We suggest that the N-terminal interaction would be a more specific and effective target in the case of prostate cancer than the LxxLL/AF2 interaction.

Carboplatin-based chemotherapy for bladder cancer.

Urothelial cancer is common and the prognosis of patients with locally advanced tumors treated with conventional treatment is poor. In this trial we examined responses among 72 urothelial cancer patients referred for treatment with MVMJ (methotrexate/vinblastine/mitoxantrone/carboplatin) chemotherapy. Sixty-four evaluable patients, 37 with locally advanced and 27 with metastatic urothelial cancer, were treated. Twenty-nine (45%) of the 64 patients had a complete or partial response, 15 (23%) had stable disease, and 13 (20%) had disease progression. The median survival of the responding patients has not been reached (range, 114 to 1184+ days). Twelve patients (32%) with locally advanced disease had a complete response to treatment; their median survival has not been reached and ranges up to 1131+ days. Five patients (18%) with metastatic cancer had a complete response to treatment and their median survival was 497 days (range, 184 to 637+). There were seven deaths within the first treatment month.

Second messenger up-regulation of androgen receptor gene transcription is absent in androgen insensitive human prostatic carcinoma cell lines, PC-3 and DU-145.

A theoretical pathway of transcriptional regulation of the androgen receptor (AR) gene is via a cAMP response element (CRE) present in its promoter region (-508 to -501). After 20 h of stimulation with 8-bromo-cAMP, AR mRNA was upregulated in LNCaP but not in either PC-3 or DU-145 cell lines. We have demonstrated that the level of CRE binding protein (CREB) was the same in all cell lines and that the putative AR-CRE forms specific and compatible protein interactions with CREB. The ability to regulate AR gene transcription via the second messenger pathway is lost in the PC-3 and DU-145 cell lines. This may be an important primary mechanism of androgen insensitivity in prostate cancer.

Sequential interleukin-2 and alpha interferon for renal cell carcinoma and melanoma.

There is a theoretical basis for the synergy of interleukin-2 (IL-2) with other cytokines. We have investigated sequential treatment with IL-2 and alpha interferon. 1 of 22 patients with metastatic renal cell carcinoma had a partial response and one a minimal response to continuous infusion IL-2 but none of the 9 patients with melanoma responded. 16 of 17 patients with renal cell cancer, and 8 with melanoma, were then treated with alpha interferon. 2 patients with renal cell cancer responded to alpha interferon with sustained remissions of 30 and 40 months; both had responded to IL-2. The investigation of combination therapy with other cytokines is suggested, by these unusually long responses to alpha interferon.

Palliation of malignant intestinal obstruction using octreotide.

Vomiting due to malignant intestinal obstruction is an unpleasant terminal event in many cancer patients, which responds poorly to conventional therapies. Somatostatin and its long-acting analogues reduce intestinal secretion. For this reason, octreotide was used in a phase I/II study of patients with intractable vomiting secondary to intestinal obstruction due to malignant disease. Vomiting was controlled or the volume of nasogastric aspirate was markedly reduced in 18 of 24 (75%) patients receiving a subcutaneous infusion of octreotide (median initial dose 300, range 100-600 micrograms/day) for a median of 9.4 (range 1-38) days. A further 2 patients had partial relief of their symptoms. Octreotide is an effective treatment of nausea and vomiting due to malignant bowel obstruction.