Gwo-Shu Mary Lee

Whole exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer

Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we report an integrated process to isolate, qualify and sequence whole exomes of CTCs with high fidelity using a census-based sequencing strategy. Power calculations suggest that mapping of >99.995% of the standard exome is possible in CTCs. We validated our process in two patients with prostate cancer, including one for whom we sequenced CTCs, a lymph node metastasis and nine cores of the primary tumor. Fifty-one of 73 CTC mutations (70%) were present in matched tissue. Moreover, we identified 10 early trunk and 56 metastatic trunk mutations in the non-CTC tumor samples and found 90% and 73% of these mutations, respectively, in CTC exomes. This study establishes a foundation for CTC genomics in the clinic.

The role of microRNA-221 and microRNA-222 in androgen-independent prostate cancer cell lines.

Androgen-dependent prostate cancer typically progresses to castration-resistant prostate cancer (CRPC) after the androgen deprivation therapy. MicroRNAs (miR) are noncoding small RNAs (19-25nt) that play an important role in the regulation of gene expression. Recent studies have shown that miR expression patterns are significantly different in normal and neoplastic prostate epithelial cells. However, the importance of miRs in the development of CRPC has not yet been explored. By performing genome-wide expression profiling of miRs, we found that expression levels of several miRs, in particular miR-221 and miR-222, were significantly increased in CRPC cells (the LNCaP-derived cell line LNCaP-Abl), compared with those in the androgen-dependent prostate cancer cell line (LNCaP). Overexpression of miR-221 or miR-222 in LNCaP or another androgen-dependent cell line, LAPC-4, significantly reduced the level of the dihydrotestosterone (DHT) induced up-regulation of prostate-specific antigen (PSA) expression and increased androgen-independent growth of LNCaP cells. Knocking down the expression level of miR-221 and miR-222 with antagonist miRs in the LNCaP-Abl cell line restored the response to the DHT induction of PSA transcription and also increased the growth response of the LNCaP-Abl cells to the androgen treatment. Changing the expression level of p27/kip1, a known target of miR-221 and miR-222, alone in LNCaP cells affected the DHT-independent cell growth but did not significantly influence the response of PSA transcription to the DHT treatment. In conclusion, our data suggest the involvement of miR-221 and miR-222 in the development or maintenance of the CRPC phenotype.

Expression differences of circulating microRNAs in metastatic castration resistant prostate cancer and low-risk, localized prostate cancer.

Recent studies show that microRNAs (miRNAs), small non‐coding RNAs that negatively regulate gene expression, may have potential for monitoring cancer status. We investigated circulating miRNAs in prostate cancer that may be associated with the progression of hormone‐sensitive primary tumors to metastatic castration resistant prostate cancer (CRPC) after androgen deprivation therapy.

Enhancer RNAs participate in androgen receptor-driven looping that selectively enhances gene activation

Significance We report that enhancer RNAs (eRNAs), a class of long noncoding RNAs, participate in the androgen receptor (AR)-dependent looping complex that enhances spatial communication of distal enhancers and target promoters, leading to transcriptional activation events. Furthermore, our data show that KLK3 eRNA (KLK3e) selectively enhances the gene expression of AR-regulated genes, and provide evidence for a positive regulatory loop in which AR-dependent transcription is modulated by an intermediate eRNA. These findings may translate into improved RNA-based therapy (eRNA suppression) to enhance the durability of androgen deprivation therapy (ADT) and prediction of the efficacy of ADT by measuring the enhancer-derived activity (eRNA expression) in prostate tumors. The androgen receptor (AR) is a key factor that regulates the behavior and fate of prostate cancer cells. The AR-regulated network is activated when AR binds enhancer elements and modulates specific enhancer–promoter looping. Kallikrein-related peptidase 3 (KLK3), which codes for prostate-specific antigen (PSA), is a well-known AR-regulated gene and its upstream enhancers produce bidirectional enhancer RNAs (eRNAs), termed KLK3e. Here, we demonstrate that KLK3e facilitates the spatial interaction of the KLK3 enhancer and the KLK2 promoter and enhances long-distance KLK2 transcriptional activation. KLK3e carries the core enhancer element derived from the androgen response element III (ARE III), which is required for the interaction of AR and Mediator 1 (Med1). Furthermore, we show that KLK3e processes RNA-dependent enhancer activity depending on the integrity of core enhancer elements. The transcription of KLK3e was detectable and its expression is significantly correlated with KLK3 (R2 = 0.6213, P < 5 × 10−11) and KLK2 (R2 = 0.5893, P < 5 × 10−10) in human prostate tissues. Interestingly, RNAi silencing of KLK3e resulted in a modest negative effect on prostate cancer cell proliferation. Accordingly, we report that an androgen-induced eRNA scaffolds the AR-associated protein complex that modulates chromosomal architecture and selectively enhances AR-dependent gene expression.

SLCO2B1 and SLCO1B3 May Determine Time to Progression for Patients Receiving Androgen Deprivation Therapy for Prostate Cancer

PURPOSE Androgen deprivation therapy (ADT), an important treatment for advanced prostate cancer, is highly variable in its effectiveness. We hypothesized that genetic variants of androgen transporter genes, SLCO2B1 and SLCO1B3, may determine time to progression on ADT. PATIENTS AND METHODS A cohort of 538 patients with prostate cancer treated with ADT was genotyped for SLCO2B1 and SLCO1B3 single nucleotide polymorphisms (SNP). The biologic function of a SLCO2B1 coding SNP in transporting androgen was examined through biochemical assays. RESULTS Three SNPs in SLCO2B1 were associated with time to progression (TTP) on ADT (P < .05). The differences in median TTP for each of these polymorphisms were about 10 months. The SLCO2B1 genotype, which allows more efficient import of androgen, enhances cell growth and is associated with a shorter TTP on ADT. Patients carrying both SLCO2B1 and SLCO1B3 genotypes, which import androgens more efficiently, exhibited a median 2-year shorter TTP on ADT, demonstrating a gene-gene interaction (P(interaction) = .041). CONCLUSION Genetic variants of SLCO2B1 and SLCO1B3 may function as pharmacogenomic determinants of resistance to ADT in prostate cancer.

A whole-blood RNA transcript-based prognostic model in men with castration-resistant prostate cancer: a prospective study

BACKGROUND Survival for patients with castration-resistant prostate cancer is highly variable. We assessed the effectiveness of a whole-blood RNA transcript-based model as a prognostic biomarker in castration-resistant prostate cancer. METHODS Peripheral blood was prospectively collected from 62 men with castration-resistant prostate cancer on various treatment regimens who were enrolled in a training set at the Dana-Farber Cancer Institute (Boston, MA, USA) from August, 2006, to June, 2008, and from 140 patients with castration-resistant prostate cancer in a validation set from Memorial Sloan-Kettering Cancer Center (New York, NY, USA) from August, 2006, to February, 2009. A panel of 168 inflammation-related and prostate cancer-related genes was assessed with optimised quantitative PCR to assess biomarkers predictive of survival. FINDINGS A six-gene model (consisting of ABL2, SEMA4D, ITGAL, and C1QA, TIMP1, CDKN1A) separated patients with castration-resistant prostate cancer into two risk groups: a low-risk group with a median survival of more than 34·9 months (median survival was not reached) and a high-risk group with a median survival of 7·8 months (95% CI 1·8-13·9; p<0·0001). The prognostic utility of the six-gene model was validated in an independent cohort. This model was associated with a significantly higher area under the curve compared with a clinicopathological model (0·90 [95% CI 0·78-0·96] vs 0·65 [0·52-0·78]; p=0·0067). INTERPRETATION Transcriptional profiling of whole blood yields crucial prognostic information about men with castration-resistant prostate cancer. The six-gene model suggests possible dysregulation of the immune system, a finding that warrants further study. FUNDING Source MDX.

The altered expression of MiR-221/-222 and MiR-23b/-27b is associated with the development of human castration resistant prostate cancer†‡

We have previously identified seven miRs‐miR‐221, ‐222, ‐23b, ‐27b, ‐15a, ‐16‐1, and ‐203, that are differentially expressed in the hormone sensitive LNCaP cell line and the hormone resistant LNCaP‐abl cell line and hypothesized that these miRs may characterize certain subtypes of human castration resistant prostate cancer (CRPC).

Androgen Receptor Mediates the Expression of UDP-Glucuronosyltransferase 2 B15 and B17 Genes

Enhanced androgen receptor (AR) activity by increased testosterone availability may play important roles in prostate cancer progressing to castration resistant state. Comparison of expression profiles in androgen dependent and independent prostate tumors demonstrated a marked increase of the expression of UDP‐glucuronosyltransferase 2B15 (UGT2B15), an androgen catabolic enzyme. We investigated mechanisms controlling the differential expression of UGT2B15 and B17 in response to androgen treatments.

Single nucleotide polymorphisms and risk of recurrence of renal-cell carcinoma: a cohort study

BACKGROUND Germline genetic polymorphisms might affect the risk of recurrence in patients with localised renal-cell carcinoma. We investigated the association between genetic polymorphisms and recurrence of renal-cell carcinoma. METHODS We analysed germline DNA samples extracted from patients with localised renal-cell carcinoma treated at the Dana-Farber/Harvard Cancer Center (Boston, MA, USA). We selected a discovery cohort from a prospective database at the Dana-Farber/Harvard Cancer Center and selected a validation cohort from department records at the Brigham and Womens Hospital (Boston, MA, USA). We validated the findings from the discovery cohort in the validation cohort. We genotyped 70 genes involved in the pathogenesis of renal-cell carcinoma (including the VHL/HIF/VEGF and PI3K/AKT/mTOR pathways, and genes involved in immune regulation and metabolism) for single nucleotide polymorphisms. We assessed the association between genotype and recurrence-free survival, adjusted for baseline characteristics, with the Cox proportional hazards model, the Kaplan-Meier method, and the log-rank test. We used a false discovery rate q value to adjust for multiple comparisons. FINDINGS We included 554 patients (403 in the discovery cohort and 151 in the validation cohort). We successfully genotyped 290 single nucleotide polymorphisms in the discovery cohort, but excluded five because they did not have a variant group for comparison. The polymorphism rs11762213, which causes a synonymous aminoacid change in MET (144G→A, located in exon 2), was associated with recurrence-free survival. Patients with one or two copies of the minor (risk) allele had an increased risk of recurrence or death (hazard ratio [HR] 1·86, 95% CI 1·17-2·95; p=0·0084) in multivariate analysis. Median recurrence-free survival for carriers of the risk allele was 19 months (95% CI 9-not reached) versus 50 months (95% CI 37-75) for patients without the risk allele. In the validation cohort the HR was 2·45 (95% CI 1·01-5·95; p=0·048). INTERPRETATION Patients with localised renal-cell carcinoma and the MET polymorphism rs11762213 might have an increased risk of recurrence after nephrectomy. If these results are further validated in a similar population, they could be incorporated into future prognostic instruments, potentially aiding the design of adjuvant clinical trials of MET inhibitors and management of renal-cell carcinoma. FUNDING Conquer Cancer Foundation and American Society of Clinical Oncology (Career Development Award); The Trust Family Research Fund for Kidney Cancer; US National Institutes of Health, National Cancer Institute Kidney Cancer Specialized Program of Research Excellence.

Resistance to docetaxel in prostate cancer is associated with androgen receptor activation and loss of KDM5D expression

Significance The results of the recent clinical trials, ChemoHormonal Therapy Versus Androgen Ablation Randomized Trial for Extensive Disease in Prostate Cancer (CHAARTED) and Systemic Therapy in Advanced or Metastatic Prostate Cancer: Evaluation of Drug Efficacy (STAMPEDE), suggest a significant contribution of androgen receptor signaling to sensitivity of docetaxel in prostate cancer. This study provides evidence that KDM5D (lysine-specific demethylase 5D) encoded on the Y chromosome plays an important role in docetaxel sensitivity by interacting with androgen receptor signaling, and that its expression level is associated with clinical outcomes. These data start to elucidate the biological underpinnings for the findings from these clinical trials. The androgen receptor (AR) plays an essential role in prostate cancer, and suppression of its signaling with androgen deprivation therapy (ADT) has been the mainstay of treatment for metastatic hormone-sensitive prostate cancer for more than 70 y. Chemotherapy has been reserved for metastatic castration-resistant prostate cancer (mCRPC). The Eastern Cooperative Oncology Group-led trial E3805: ChemoHormonal Therapy Versus Androgen Ablation Randomized Trial for Extensive Disease in Prostate Cancer (CHAARTED) showed that the addition of docetaxel to ADT prolonged overall survival compared with ADT alone in patients with metastatic hormone-sensitive prostate cancer. This finding suggests that there is an interaction between AR signaling activity and docetaxel sensitivity. Here we demonstrate that the prostate cancer cell lines LNCaP and LAPC4 display markedly different sensitivity to docetaxel with AR activation, and RNA-seq analysis of these cell lines identified KDM5D (lysine-specific demethylase 5D) encoded on the Y chromosome as a potential mediator of this sensitivity. Knocking down KDM5D expression in LNCaP leads to docetaxel resistance in the presence of dihydrotestosterone. KDM5D physically interacts with AR in the nucleus, and regulates its transcriptional activity by demethylating H3K4me3 active transcriptional marks. Attenuating KDM5D expression dysregulates AR signaling, resulting in docetaxel insensitivity. KDM5D deletion was also observed in the LNCaP-derived CRPC cell line 104R2, which displayed docetaxel insensitivity with AR activation, unlike parental LNCaP. Dataset analysis from the Oncomine database revealed significantly decreased KDM5D expression in CRPC and poorer prognosis with low KDM5D expression. Taking these data together, this work indicates that KDM5D modulates the AR axis and that this is associated with altered docetaxel sensitivity.