Antiangiogenic therapy resistance occurs frequently in patients with metastatic renal cell carcinoma (RCC). The purpose of this study was to understand the mechanism of resistance to sunitinib, an antiangiogenic small molecule, and to exploit this mechanism therapeutically. We hypothesized that sunitinib-induced upregulation of the prometastatic MET and AXL receptors is associated with resistance to sunitinib and with more aggressive tumor behavior. In the present study, tissue microarrays containing sunitinib treated and untreated RCC tissues were stained with MET and AXL antibodies. The low malignant RCC cell line, 786-O, was chronically treated with sunitinib, and assayed for AXL, MET, epithelial mesenchymal transition (EMT) protein expression and activation. Co-culture experiments were used to examine the effect of sunitinib pretreatment on endothelial cell growth. The effects of AXL and MET were evaluated in various cell-based models by shRNA or inhibition by cabozantinib, the multi-tyrosine kinases inhibitor that targets VEGFR, MET and AXL. Xenograft mouse models tested the ability of cabozantinib to rescue sunitinib resistance. We demonstrated that increased AXL and MET expression was associated with inferior clinical outcome in patients. Chronic sunitinib treatment of RCC cell lines activated both AXL and MET, induced EMT associated gene expression changes including upregulation of Snail and β-catenin, and increased cell migration and invasion. Pretreatment with sunitinib enhanced angiogenesis in 786-0/HUVEC co-culture models. The suppression of AXL or MET expression, and the inhibition of AXL and MET activation using cabozantinib both impaired chronic sunitinib treatment-induced prometastatic behavior in cell culture, and rescued acquired resistance to sunitinib in xenograft models. In summary, chronic sunitinib treatment induces the activation of AXL and MET signaling and promotes pro-metastatic behavior and angiogenesis. The inhibition of AXL and MET activity may overcome resistance induced by prolonged sunitinib therapy in metastatic RCC.

Background Enzalutamide is a novel antiandrogen with proven efficacy in metastatic castration-resistant prostate cancer (mCRPC). Objective To evaluate enzalutamide’s effects on cancer and on androgens in blood and bone marrow, and associate these with clinical observations. Design, setting, and participants In this prospective phase 2 study, 60 patients with bone mCRPC received enzalutamide 160 mg orally daily and had transilial bone marrow biopsies before treatment and at 8 wk of treatment. Outcome measurements and statistical analysis Androgen signaling components (androgen receptor [AR], ARV7, v-ets avian erythroblastosis virus E26 oncogene homolog [ERG], cytochrome P450, family 17, subfamily A, polypeptide 1 [CYP17]) and molecules implicated in mCRPC progression (phospho-Met, phospho-Src, glucocorticoid receptor, Ki67) were assessed by immunohistochemistry; testosterone, cortisol, and androstenedione concentrations were assessed by liquid chromatography–tandem mass spectrometry; and AR copy number was assessed by real-time polymerase chain reaction. Descriptive statistics were applied. Results and limitations Median time to treatment discontinuation was 22 wk (95% confidence interval, 19.9–29.6). Twenty-two (37%) patients exhibited primary resistance to enzalutamide, discontinuing treatment within 4 mo. Maximal prostate-specific antigen (PSA) decline ≥50% and ≥90% occurred in 27 (45%) and 13 (22%) patients, respectively. Following 8 wk of treatment, bone marrow and circulating testosterone levels increased. Pretreatment tumor nuclear AR overexpression (>75%) and CYP17 (>10%) expression were associated with benefit (p = 0.018). AR subcellular localization shift from the nucleus was confirmed in eight paired samples (with PSA decline) of 23 evaluable paired samples. Presence of an ARV7 variant was associated with primary resistance to enzalutamide (p = 0.018). Limited patient numbers warrant further validation. Conclusions The observed subcellular shift of AR from the nucleus and increased testosterone concentration provide the first evidence in humans that enzalutamide suppresses AR signaling while inducing an adaptive feedback. Persistent androgen signaling in mCRPC was predictive of benefit and ARV7 was associated with primary resistance. Patient summary We report a first bone biopsy study in metastatic prostate cancer in humans that searched for predictors of outcome of enzalutamide therapy. Benefit is linked to a pretreatment androgen-signaling signature.

Prostate cancer antigen 3 (PCA3) is the most specific prostate cancer biomarker but its function remains unknown. Here we identify PRUNE2, a target protein-coding gene variant, which harbors the PCA3 locus, thereby classifying PCA3 as an antisense intronic long noncoding (lnc)RNA. We show that PCA3 controls PRUNE2 levels via a unique regulatory mechanism involving formation of a PRUNE2/PCA3 double-stranded RNA that undergoes adenosine deaminase acting on RNA (ADAR)-dependent adenosine-to-inosine RNA editing. PRUNE2 expression or silencing in prostate cancer cells decreased and increased cell proliferation, respectively. Moreover, PRUNE2 and PCA3 elicited opposite effects on tumor growth in immunodeficient tumor-bearing mice. Coregulation and RNA editing of PRUNE2 and PCA3 were confirmed in human prostate cancer specimens, supporting the medical relevance of our findings. These results establish PCA3 as a dominant-negative oncogene and PRUNE2 as an unrecognized tumor suppressor gene in human prostate cancer, and their regulatory axis represents a unique molecular target for diagnostic and therapeutic intervention.S everal lines of evidence demonstrate that long noncoding RNAs (lncRNAs) are functional in carcinogenesis through regulatory mechanisms such as promoter looping, alternative splicing, antisense gene silencing, transcriptional regulation, and DNA repair, thus potentially serving as tumor markers. A few lncRNA species have emerged as potential prostate cancer biomarkers such as prostate cancer gene expression marker-1 (PCGEM1) and prostate cancer noncoding RNA1 (PRNCR1), which enhance androgen receptor (AR)-dependent gene activation, and prostate cancer-associated ncRNA transcript-1 (PCAT1), which silences BRCA2 via posttranscriptional homologous recombination (1). Notably, the most specific biomarker in human prostate cancer identified to date is an lncRNA, prostate cancer antigen 3 (PCA3, also known as PCA3 DD3 or DD3 PCA3 ), which is up-regulated in human prostate cancer (2). Since its discovery more than 15 y ago, PCA3 has been extensively investigated (3) and has been approved for clinical applications to aid the diagnosis of prostate cancer in both the European Union and the United States. Paradoxically-despite its striking clinical specificity-the inherent cellular role of the lncRNA PCA3 in human prostate cancer, if any, remains completely unknown (1). Here we report a unique biological function for PCA3. Within a single functional genetic unit, we show that PCA3 is an antisense intronic lncRNA that down-regulates an as yet unrecognized tumor suppressor gene, a human homolog of the Drosophila prune gene, PRUNE2, through a process that involves RNA editing mediated by a supramolecular complex containing adenosine deaminase acting on RNA (ADAR) family members. We propose a working model in which PCA3 acts as a dominant-negative oncogene in prostate cancer and show consistent results in therapeutic preclinical models and in patient-derived human samples. Therefore, the molecular interaction of PRUNE2...

A B S T R A C T PurposePersistent androgen signaling is implicated in castrate-resistant prostate cancer (CRPC) progression. This study aimed to evaluate androgen signaling in bone marrow-infiltrating cancer and testosterone in blood and bone marrow and to correlate with clinical observations. Patients and MethodsThis was an open-label, observational study of 57 patients with bone-metastatic CRPC who underwent transiliac bone marrow biopsy between October 2007 and March 2010. Patients received oral abiraterone acetate (1 g) once daily and prednisone (5 mg) twice daily. Androgen receptor (AR) and CYP17 expression were assessed by immunohistochemistry, testosterone concentration by mass spectrometry, AR copy number by polymerase chain reaction, and TMPRSS2-ERG status by fluorescent in situ hybridization in available tissues. ResultsMedian overall survival was 555 days (95% CI, 440 to 965ϩ days). Maximal prostate-specific antigen decline Ն 50% occurred in 28 (50%) of 56 patients. Homogeneous, intense nuclear expression of AR, combined with Ն 10% CYP17 tumor expression, was correlated with longer time to treatment discontinuation (Ͼ 4 months) in 25 patients with tumor-infiltrated bone marrow samples. Pretreatment CYP17 tumor expression Ն 10% was correlated with increased bone marrow aspirate testosterone. Blood and bone marrow aspirate testosterone concentrations declined to less than picograms-per-milliliter levels and remained suppressed at progression. ConclusionThe observed pretreatment androgen-signaling signature is consistent with persistent androgen signaling in CRPC bone metastases. This is the first evidence that abiraterone acetate achieves sustained suppression of testosterone in both blood and bone marrow aspirate to less than picograms-per-milliliter levels. Potential admixture of blood with bone marrow aspirate limits our ability to determine the origin of measured testosterone. J Clin