The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature ("Core" OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.

Nuclear iASPP may facilitate prostate cancer progression

One of the major challenges in prostate cancer (PCa) research is the identification of key players that control the progression of primary cancers to invasive and metastatic disease. The majority of metastatic PCa express wild-type p53, whereas loss of p63 expression, a p53 family member, is a common event. Here we identify inhibitor of apoptosis-stimulating protein of p53 (iASPP), a common cellular regulator of p53 and p63, as an important player of PCa progression. Detailed analysis of the prostate epithelium of iASPP transgenic mice, iASPP(Δ8/Δ8) mice, revealed that iASPP deficiency resulted in a reduction in the number of p63 expressing basal epithelial cells compared with that seen in wild-type mice. Nuclear and cytoplasmic iASPP expression was greater in PCa samples compared with benign epithelium. Importantly nuclear iASPP associated with p53 accumulation in vitro and in vivo. A pair of isogenic primary and metastatic PCa cell lines revealed that nuclear iASPP is enriched in the highly metastatic PCa cells. Nuclear iASPP is often detected in PCa cells located at the invasive leading edge in vivo. Increased iASPP expression associated with metastatic disease and PCa-specific death in a clinical cohort with long-term follow-up. These results suggest that iASPP function is required to maintain the expression of p63 in normal basal prostate epithelium, and nuclear iASPP may inactivate p53 function and facilitate PCa progression. Thus iASPP expression may act as a predictive marker of PCa progression.

Natural history of surgically treated high-risk prostate cancer

BACKGROUND No data exist on the patterns of biochemical recurrence (BCR) and their effect on survival in patients with high-risk prostate cancer (PCa) treated with surgery. The aim of our investigation was to evaluate the natural history of PCa in patients treated with radical prostatectomy (RP) alone. MATERIALS AND METHODS Overall, 2,065 patients with high-risk PCa treated with RP at 7 tertiary referral centers between 1991 and 2011 were identified. First, we calculated the probability of experiencing BCR after surgery. Particularly, we relied on conditional survival estimates for BCR after RP. Competing-risks regression analyses were then used to evaluate the effect of time to BCR on the risk of cancer-specific mortality (CSM). RESULTS Median follow-up was 70 months. Overall, the 5-year BCR-free survival rate was 55.2%. Given the BCR-free survivorship at 1, 2, 3, 4, and 5 years, the BCR-free survival rates improved by+7.6%,+4.1%,+4.8%,+3.2%, and+3.7%, respectively. Overall, the 10-year CSM rate was 14.8%. When patients were stratified according to time to BCR, patients experiencing BCR within 36 months from surgery had higher 10-year CSM rates compared with those experiencing late BCR (19.1% vs. 4.4%; P<0.001). At multivariate analyses, time to BCR represented an independent predictor of CSM (P<0.001). CONCLUSIONS Increasing time from surgery is associated with a reduction of the risk of subsequent BCR. Additionally, time to BCR represents a predictor of CSM in these patients. These results might help provide clinicians with better follow-up strategies and more aggressive treatments for early BCR.

Loss of tumor suppressor mir-203 mediates overexpression of LIM and SH3 Protein 1 (LASP1) in high-risk prostate cancer thereby increasing cell proliferation and migration

Several studies have linked overexpression of the LIM and SH3 domain protein 1 (LASP1) to progression of breast, colon, liver, and bladder cancer. However, its expression pattern and role in human prostate cancer (PCa) remained largely undefined. Analysis of published microarray data revealed a significant overexpression of LASP1 in PCa metastases compared to parental primary tumors and normal prostate epithelial cells. Subsequent gene-set enrichment analysis comparing LASP1-high and -low PCa identified an association of LASP1 with genes involved in locomotory behavior and chemokine signaling. These bioinformatic predictions were confirmed in vitro as the inducible short hairpin RNA-mediated LASP1 knockdown impaired migration and proliferation in LNCaP prostate cancer cells. By immunohistochemical staining and semi-quantitative image analysis of whole tissue sections we found an enhanced expression of LASP1 in primary PCa and lymph node metastases over benign prostatic hyperplasia. Strong cytosolic and nuclear LASP1 immunoreactivity correlated with PSA progression. Conversely, qRT-PCR analyses for mir-203, which is a known translational suppressor of LASP1 in matched RNA samples revealed an inverse correlation of LASP1 protein and mir-203 expression. Collectively, our results suggest that loss of mir-203 expression and thus uncontrolled LASP1 overexpression might drive progression of PCa.

Methylation of PITX2, HOXD3, RASSF1 and TDRD1 predicts biochemical recurrence in high-risk prostate cancer

PURPOSE To explore differential methylation of HAAO, HOXD3, LGALS3, PITX2, RASSF1 and TDRD1 as a molecular tool to predict biochemical recurrence (BCR) in patients with high-risk prostate cancer (PCa). METHODS A multiplexed nested methylation-specific PCR was applied to quantify promoter methylation of the selected markers in five cell lines, 42 benign prostatic hyperplasia (BPH) and 71 high-risk PCa tumor samples. Uni- and multivariate Cox regression models were used to assess the importance of the methylation level in predicting BCR. RESULTS A PCa-specific methylation marker HAAO in combination with HOXD3 and a hypomethylation marker TDRD1 distinguished PCa samples (>90 % of tumor cells each) from BPH with a sensitivity of 0.99 and a specificity of 0.95. High methylation of PITX2, HOXD3 and RASSF1, as well as low methylation of TDRD1, appeared to be significantly associated with a higher risk for BCR (HR 3.96, 3.44, 2.80 and 2.85, correspondingly) after correcting for established risk factors. When DNA methylation was treated as a continuous variable, a two-gene model PITX2 × 0.020677 + HOXD3 × 0.0043132 proved to be the best predictor of BCR (HR 4.85) compared with the individual markers. This finding was confirmed in an independent set of 52 high-risk PCa tumor samples (HR 11.89). CONCLUSIONS Differential promoter methylation of HOXD3, PITX2, RASSF1 and TDRD1 emerges as an independent predictor of BCR in high-risk PCa patients. A two-gene continuous DNA methylation model "PITX2 × 0.020677 + HOXD3 × 0.0043132" is a better predictor of BCR compared with individual markers.

Survival in patients with high-risk prostate cancer is predicted by miR-221, which regulates proliferation, apoptosis, and invasion of prostate cancer cells by inhibiting IRF2 and SOCS3

A lack of reliably informative biomarkers to distinguish indolent and lethal prostate cancer is one reason this disease is overtreated. miR-221 has been suggested as a biomarker in high-risk prostate cancer, but there is insufficient evidence of its potential utility. Here we report that miR-221 is an independent predictor for cancer-related death, extending and validating earlier findings. By mechanistic investigations we showed that miR-221 regulates cell growth, invasiveness, and apoptosis in prostate cancer at least partially via STAT1/STAT3-mediated activation of the JAK/STAT signaling pathway. miR-221 directly inhibits the expression of SOCS3 and IRF2, two oncogenes that negatively regulate this signaling pathway. miR-221 expression sensitized prostate cancer cells for IFN-γ-mediated growth inhibition. Our findings suggest that miR-221 offers a novel prognostic biomarker and therapeutic target in high-risk prostate cancer.

Stratification of high-risk prostate cancer into prognostic categories: a European multi-institutional study

BACKGROUND High-risk prostate cancer (PCa) is an extremely heterogeneous disease. A clear definition of prognostic subgroups is mandatory. OBJECTIVE To develop a pretreatment prognostic model for PCa-specific survival (PCSS) in high-risk PCa based on combinations of unfavorable risk factors. DESIGN, SETTING, AND PARTICIPANTS We conducted a retrospective multicenter cohort study including 1360 consecutive patients with high-risk PCa treated at eight European high-volume centers. INTERVENTION Retropubic radical prostatectomy with pelvic lymphadenectomy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Two Cox multivariable regression models were constructed to predict PCSS as a function of dichotomization of clinical stage (< cT3 vs cT3-4), Gleason score (GS) (2-7 vs 8-10), and prostate-specific antigen (PSA; ≤ 20 ng/ml vs > 20 ng/ml). The first "extended" model includes all seven possible combinations; the second "simplified" model includes three subgroups: a good prognosis subgroup (one single high-risk factor); an intermediate prognosis subgroup (PSA >20 ng/ml and stage cT3-4); and a poor prognosis subgroup (GS 8-10 in combination with at least one other high-risk factor). The predictive accuracy of the models was summarized and compared. Survival estimates and clinical and pathologic outcomes were compared between the three subgroups. RESULTS AND LIMITATIONS The simplified model yielded an R(2) of 33% with a 5-yr area under the curve (AUC) of 0.70 with no significant loss of predictive accuracy compared with the extended model (R(2): 34%; AUC: 0.71). The 5- and 10-yr PCSS rates were 98.7% and 95.4%, 96.5% and 88.3%, 88.8% and 79.7%, for the good, intermediate, and poor prognosis subgroups, respectively (p = 0.0003). Overall survival, clinical progression-free survival, and histopathologic outcomes significantly worsened in a stepwise fashion from the good to the poor prognosis subgroups. Limitations of the study are the retrospective design and the long study period. CONCLUSIONS This study presents an intuitive and easy-to-use stratification of high-risk PCa into three prognostic subgroups. The model is useful for counseling and decision making in the pretreatment setting.

Myoglobin expression in prostate cancer is correlated to androgen receptor expression and markers of tumor hypoxia.

Recent studies identified unexpected expression and transcriptional complexity of the hemoprotein myoglobin (MB) in human breast cancer but its role in prostate cancer is still unclear. Expression of MB was immunohistochemically analyzed in three independent cohorts of radical prostatectomy specimens (n = 409, n = 625, and n = 237). MB expression data were correlated with clinicopathological parameters and molecular parameters of androgen and hypoxia signaling. Expression levels of novel tumor-associated MB transcript variants and the VEGF gene as a hypoxia marker were analyzed using qRT-PCR. Fifty-three percent of the prostate cancer cases were MB positive and significantly correlated with androgen receptor (AR) expression (p < 0.001). The positive correlation with CAIX (p < 0.001) and FASN (p = 0.008) as well as the paralleled increased expression of the tumor-associated MB transcript variants and VEGF suggest that hypoxia participates in MB expression regulation. Analogous to breast cancer, MB expression in prostate cancer is associated with steroid hormone signaling and markers of hypoxia. Further studies must elucidate the novel functional roles of MB in human carcinomas, which probably extend beyond its classic intramuscular function in oxygen storage.

Intratumoral hypoxia as the genesis of genetic instability and clinical prognosis in prostate cancer

Intratumoral hypoxia is prevalent in many solid tumors and is a marker of poor clinical prognosis in prostate cancer. The presence of hypoxia is associated with increased chromosomal instability, gene amplification, downregulation of DNA damage repair pathways, and altered sensitivity to agents that damage DNA. These genomic changes could also lead to oncogene activation or tumor suppressor gene inactivation during prostate cancer progression. We review here the concept of repair-deficient hypoxic tumor cells that can adapt to low oxygen levels and acquire an aggressive "unstable mutator" phenotype. We speculate that hypoxia-induced genomic instability may also be a consequence of aberrant mitotic function in hypoxic cells, which leads to increased chromosomal instability and aneuploidy. Because both hypoxia and aneuploidy are prognostic factors in prostate cancer, a greater understanding of these biological states in prostate cancer may lead to novel prognostic and predictive tests and drive new therapeutic strategies in the context of personalized cancer medicine.

DNA Methylation-Guided Prediction of Clinical Failure in High-Risk Prostate Cancer.

BACKGROUND Prostate cancer (PCa) is a very heterogeneous disease with respect to clinical outcome. This study explored differential DNA methylation in a priori selected genes to diagnose PCa and predict clinical failure (CF) in high-risk patients. METHODS A quantitative multiplex, methylation-specific PCR assay was developed to assess promoter methylation of the APC, CCND2, GSTP1, PTGS2 and RARB genes in formalin-fixed, paraffin-embedded tissue samples from 42 patients with benign prostatic hyperplasia and radical prostatectomy specimens of patients with high-risk PCa, encompassing training and validation cohorts of 147 and 71 patients, respectively. Log-rank tests, univariate and multivariate Cox models were used to investigate the prognostic value of the DNA methylation. RESULTS Hypermethylation of APC, CCND2, GSTP1, PTGS2 and RARB was highly cancer-specific. However, only GSTP1 methylation was significantly associated with CF in both independent high-risk PCa cohorts. Importantly, trichotomization into low, moderate and high GSTP1 methylation level subgroups was highly predictive for CF. Patients with either a low or high GSTP1 methylation level, as compared to the moderate methylation groups, were at a higher risk for CF in both the training (Hazard ratio [HR], 3.65; 95% CI, 1.65 to 8.07) and validation sets (HR, 4.27; 95% CI, 1.03 to 17.72) as well as in the combined cohort (HR, 2.74; 95% CI, 1.42 to 5.27) in multivariate analysis. CONCLUSIONS Classification of primary high-risk tumors into three subtypes based on DNA methylation can be combined with clinico-pathological parameters for a more informative risk-stratification of these PCa patients.

Copper-64 Labeled Macrobicyclic Sarcophagine Coupled to a GRP Receptor Antagonist Shows Great Promise for PET Imaging of Prostate Cancer

The gastrin-releasing peptide receptor (GRPr) is an important molecular target for the visualization and therapy of tumors and can be targeted with radiolabeled bombesin derivatives. The present study aims to develop statine-based bombesin receptor antagonists suitable for labeling with 64Cu for imaging by positron emission tomography (PET). The potent GRPr antagonist D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 was conjugated to the sarcophagine (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane=Sar) derivative 5-(8-methyl-3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid (MeCOSar) via PEG4 (LE1) and PEG2 (LE2) spacers and radiolabeled with 64Cu2+ with >95% yield and specific activities of about 100 MBq/nmol. Both Cu(II) conjugates have high affinity for GRPr (IC50: natCu-LE1, 1.4±0.1 nM; natCu-LE2, 3.8±0.6 nM). The antagonistic properties of both conjugates were confirmed by Ca2+-flux measurements. Biodistribution studies of Cu-64-LE1 exhibited specific targeting of the tumor (19.6±4.7% IA/g at 1 h p.i.) and GRPr-positive organs. Biodistribution and PET images at 4 and 24 h postinjection showed increasing tumor-to-background ratios with time. This was illustrated by the acquisition of PET images showing high tumor-to-normal tissue contrast. This study demonstrates the high affinity of the MeCOSar-PEGx-bombesin conjugates to GRPr. The stability of 64Cu complexes of MeCOSar, the long half-life of 64Cu, and the suitable biodistribution profile of the 64Cu-labeled peptides lead to PET images of high contrast suitable for potential translation into the clinic.

Very long-term survival patterns of young patients treated with radical prostatectomy for high-risk prostate cancer

OBJECTIVE In patients with a long life expectancy with high-risk (HR) prostate cancer (PCa), the chance to die from PCa is not negligible and may change significantly according to the time elapsed from surgery. The aim of this study was to evaluate long-term survival patterns in young patients treated with radical prostatectomy (RP) for HRPCa. MATERIALS AND METHODS Within a multiinstitutional cohort, 600 young patients (≤59 years) treated with RP between 1987 and 2012 for HRPCa (defined as at least one of the following adverse characteristics: prostate specific antigen>20, cT3 or higher, biopsy Gleason sum 8-10) were identified. Smoothed cumulative incidence plot was performed to assess cancer-specific mortality (CSM) and other cause mortality (OCM) rates at 10, 15, and 20 years after RP. The same analyses were performed to assess the 5-year probability of CSM and OCM in patients who survived 5, 10, and 15 years after RP. A multivariable competing risk regression model was fitted to identify predictors of CSM and OCM. RESULTS The 10-, 15- and 20-year CSM and OCM rates were 11.6% and 5.5% vs. 15.5% and 13.5% vs. 18.4% and 19.3%, respectively. The 5-year probability of CSM and OCM rates among patients who survived at 5, 10, and 15 years after RP, were 6.4% and 2.7% vs. 4.6% and 9.6% vs. 4.2% and 8.2%, respectively. Year of surgery, pathological stage and Gleason score, surgical margin status and lymph node invasion were the major determinants of CSM (all P≤0.03). Conversely, none of the covariates was significantly associated with OCM (all P≥ 0.09). CONCLUSIONS Very long-term cancer control in young high-risk patients after RP is highly satisfactory. The probability of dying from PCa in young patients is the leading cause of death during the first 10 years of survivorship after RP. Thereafter, mortality not related to PCa became the main cause of death. Consequently, surgery should be consider among young patients with high-risk disease and strict PCa follow-up should enforce during the first 10 years of survivorship after RP.