Histone methyltransferase DOT1L coordinates AR and MYC stability in prostate cancer.

The histone methyltransferase DOT1L methylates lysine 79 (K79) on histone H3 and is involved in Mixed Lineage Leukemia (MLL) fusion leukemogenesis; however, its role in prostate cancer (PCa) is undefined. Here we show that DOT1L is overexpressed in PCa and is associated with poor outcome. Genetic and chemical inhibition of DOT1L selectively impaired the viability of androgen receptor (AR)-positive PCa cells and organoids, including castration-resistant and enzalutamide-resistant cells. The sensitivity of AR-positive cells is due to a distal K79 methylation-marked enhancer in the MYC gene bound by AR and DOT1L not present in AR-negative cells. DOT1L inhibition leads to reduced MYC expression and upregulation of MYC-regulated E3 ubiquitin ligases HECTD4 and MYCBP2, which promote AR and MYC degradation. This leads to further repression of MYC in a negative feed forward manner. Thus DOT1L selectively regulates the tumorigenicity of AR-positive prostate cancer cells and is a promising therapeutic target for PCa.

Acquired Resistance to Poly (ADP-ribose) Polymerase Inhibitor Olaparib in BRCA2-Associated Prostate Cancer Resulting From Biallelic BRCA2 Reversion Mutations Restores Both Germline and Somatic Loss-of-Function Mutations.

PARP1/2 inhibitors are effective against BRCA2-deficient tumors. The PARP inhibitor (PARPi) olaparib received FDA breakthrough designation for treatment of metastatic castration-resistant prostate cancers (CRPC) carrying mutations in BRCA1/2 or ATM genes. Emergent resistance to PARPi has been associated with tumor-specific BRCA2 mutations that revert the normal open reading frame rescuing homologous recombination. We describe a case of metastatic CRPC with germline BRCA2 mutation with acquired resistance to olaparib related to biallelic BRCA2 reversion mutations of both the germline and somatic loss of function alleles detected by circulating tumor DNA testing. We also summarize a retrospective analysis of 1,534 prostate cancer cases with ctDNA analysis showing a 1.6% incidence of germline BRCA2 mutations. Within the germline BRCA2-positive cases exposed to platinum chemotherapy or PARP inhibition, the prevalence of reversion mutations was 40%. This report documents the frequency of reversion mutations in a large cohort of prostate cancer patients carrying of BRCA mutations. It also shows the potential utility of ctDNA analyses for early detection of reversion mutation driving tumor resistance.

Selective COX-2 inhibitor (celecoxib) decreases cellular growth in prostate cancer cell lines independent of p53.

Celecoxib is a clinically available COX-2 inhibitor that has been reported to have antineoplastic activity. It has been proposed as a preventative agent for several types of early neoplastic lesions. Earlier studies have shown that sensitivity of prostatic carcinoma (PCa) to celecoxib is associated with apoptosis; however, these studies have not demonstrated adequately whether this effect is dependent on p53 status. We studied the relation between sensitivity to celecoxib and the phenotypic p53 status of PCa cells lines, LNCaP (wild type p53), PC3 (null p53) and DU145 (mutated p53). Cellular growth was assessed at 24, 48, 72 and 96 h after celecoxib treatment at concentrations of 0, 10, 30, 50, 70 and 100 µM using an MTT assay. Cellular proliferation (Ki-67 expression) was determined by immunocytochemistry. Phenotypic expression of p53 was analyzed by western blotting. The effects of celecoxib on cellular growth and its association with p53 were assessed after down-regulation of p53 using synthetic interfering RNAs (siRNA) in LNCaP cells. Expression of p53 and COX-2 at mRNA levels was assessed by quantitative real time polymerase reaction (qRT-PCR). We found that celecoxib inhibited cellular growth and proliferation in a dose-dependent manner in all three cell lines; LNCaP cells with a native p53 were the most sensitive to celecoxib. We observed a down- regulation effect on p53 in LNCaP cells exposed to ≥ 30 µM celecoxib for 72 h, but found no significant changes in the p53 levels of DU145 cells, which have a mutated p53. Reduced COX-2 expression was found with decreased p53 in LNCaP and PC-3 cells that were exposed to ≥ 20 µM of celecoxib for 72 h, but COX-2 expression was increased in DU145 cells. All three cell lines demonstrated pan-cytotoxicity when exposed to 100 µM celecoxib. When p53 expression was inhibited using siRNA in LNCaP cells, the inhibitory effects on cellular growth usually exerted by celecoxib were not changed significantly. Celecoxib reduces the growth of prostate cancer cell lines in part by decreasing proliferation, which suggests that the inhibition of growth of LNCaP cells by celecoxib is independent of normal levels of native p53.

A mouse model of heterogeneous, c-MYC-initiated prostate cancer with loss of Pten and p53.

Human tumors are heterogeneous and evolve through a dynamic process of genetic mutation and selection. During this process, the effects of a specific mutation on the incipient cancer cell may dictate the nature of subsequent mutations that can be tolerated or selected for, affecting the rate at which subsequent mutations occur. Here we have used a new mouse model of prostate cancer that recapitulates several salient features of the human disease to examine the relative rates in which the remaining wild-type alleles of Pten and p53 tumor suppressor genes are lost. In this model, focal overexpression of c-MYC in a few prostate luminal epithelial cells provokes a mild proliferative response. In the context of compound Pten/p53 heterozygosity, c-MYC-initiated cells progress to prostatic intraepithelial neoplasia (mPIN) and adenocarcinoma lesions with marked heterogeneity within the same prostate glands. Using laser capture microdissection and gene copy number analyses, we found that the frequency of Pten loss was significantly higher than that of p53 loss in mPIN but not invasive carcinoma lesions. c-MYC overexpression, unlike Pten loss, did not activate the p53 pathway in transgenic mouse prostate cells, explaining the lack of selective pressure to lose p53 in the c-MYC-overexpressing cells. This model of heterogeneous prostate cancer based on alterations in genes relevant to the human disease may be useful for understanding pathogenesis of the disease and testing new therapeutic agents.

Pim1 kinase is required to maintain tumorigenicity in MYC-expressing prostate cancer cells.

PIM1 kinase and MYC are commonly co-expressed in human prostate cancer and synergize to induce rapidly progressing prostate cancer in mouse models. Deficiency of the Pim kinase genes is well tolerated in vivo, suggesting that PIM1 inhibition might offer an attractive therapeutic modality for prostate cancer, particularly for MYC-expressing tumors. Here we examine the molecular consequences of Pim1 and MYC overexpression in the prostate as well as the effects of depleting Pim1 in prostate carcinoma cells with high levels of MYC. Overexpression of Pim1 in the mouse prostate induces several pro-tumorigenic genetic programs including cell cycle genes and Myc-regulated genes before the induction of any discernible pathology. Pim1 depletion by RNA interference in mouse and human prostate cancer cells decreased cellular proliferation, survival, Erk signaling and tumorigenicity even when MYC levels were not significantly altered. These results indicate that PIM1 may be necessary to maintain tumorigenicity, and further support efforts aimed at developing PIM1 inhibitors for prostate cancer therapy.

Pim1 kinase synergizes with c-MYC to induce advanced prostate carcinoma.

The oncogenic PIM1 kinase has been implicated as a cofactor for c-MYC in prostate carcinogenesis. In this study, we show that in human prostate tumors, coexpression of c-MYC and PIM1 is associated with higher Gleason grades. Using a tissue recombination model coupled with lentiviral-mediated gene transfer we find that Pim1 is weakly oncogenic in naive adult mouse prostatic epithelium. However, it cooperates dramatically with c-MYC to induce prostate cancer within 6-weeks. Importantly, c-MYC/Pim1 synergy is critically dependent on Pim1 kinase activity. c-MYC/Pim1 tumors showed increased levels of the active serine-62 (S62) phosphorylated form of c-MYC. Grafts expressing a phosphomimetic c-MYCS62D mutant had higher rates of proliferation than grafts expressing wild type c-MYC but did not form tumors like c-MYC/Pim1 grafts, indicating that Pim1 cooperativity with c-MYC in vivo involves additional mechanisms other than enhancement of c-MYC activity by S62 phosphorylation. c-MYC/Pim1-induced prostate carcinomas show evidence of neuroendocrine (NE) differentiation. Additional studies, including the identification of tumor cells coexpressing androgen receptor and NE cell markers synaptophysin and Ascl1 suggested that NE tumors arose from adenocarcinoma cells through transdifferentiation. These results directly show functional cooperativity between c-MYC and PIM1 in prostate tumorigenesis in vivo and support efforts for targeting PIM1 in prostate cancer.

Frequent and early loss of the EGR1 corepressor NAB2 in human prostate carcinoma.

The transcription factor EGR1 is frequently overexpressed in human prostate cancer and regulates the expression of several genes important for tumor progression. In addition, mice lacking the Egr1 gene show a defect in prostate tumorigenesis. NAB2 is a novel corepressor molecule that modulates EGR1 activity and is induced by the same stimuli that induce EGR1. The human NAB2 gene has been localized to 12q13.3-14.1, within a chromosomal region that is thought to harbor a prostate tumor suppressor. We have examined the expression of NAB2 in human prostate carcinoma specimens. We show here that NAB2 protein expression is lost in a majority of primary prostate carcinoma specimens, including many samples that have high EGR1 levels. This loss occurs early in the tumorigenic process and is sustained, as it is seen in precursor prostatic intraepithelial neoplasia lesions as well as in metastases. Furthermore, loss of NAB2 did not correlate with the tumor grade or stage. Our findings suggest that high levels of EGR1 coupled with low levels of NAB2 can result in high, unrestrained EGR1 transcriptional activity in human prostate cancers.

Impaired prostate tumorigenesis in Egr1-deficient mice.

The transcription factor early growth response protein 1 (EGR1) is overexpressed in a majority of human prostate cancers and is implicated in the regulation of several genes important for prostate tumor progression. Here we have assessed the effect of Egr1 deficiency on tumor development in two transgenic mouse models of prostate cancer (CR2-T-Ag and TRAMP). Using a combination of high-resolution magnetic resonance imaging and histopathological and survival analyses, we show that tumor progression was significantly impaired in Egr1-/- mice. Tumor initiation and tumor growth rate were not affected by the lack of Egr1; however, Egr1 deficiency significantly delayed the progression from prostatic intra-epithelial neoplasia to invasive carcinoma. These results indicate a unique role for Egr1 in regulating the transition from localized, carcinoma in situ to invasive carcinoma.

EGR1 target genes in prostate carcinoma cells identified by microarray analysis.

The EGR1 transactivator is overexpressed in prostate cancer, and its expression pattern suggests that EGR1 could potentially regulate a number of steps involved in initiation and progression of prostate cancer, such as mitogenesis, invasiveness, angiogenesis, and metastasis. To identify potential EGR1 target genes in an unbiased manner, we have utilized adenovirus-mediated expression of EGR1 in a prostate cancer cell line to identify specific genes that are induced by EGR1. Using oligonucleotide arrays, a number of EGR1-regulated genes were identified and their regulation was confirmed by quantitative reverse transcription-polymerase chain reaction analysis. One of the largest gene classes identified in this screen includes several neuroendocrine-associated genes (neuron-specific enolase, neurogranin), suggesting that EGR1 overexpression may contribute to the neuroendocrine differentiation that often accompanies prostate cancer progression. This screen also identified several growth factors such as insulin-like growth factor-II, platelet-derived growth factor-A, and transforming growth factor-beta1, which have previously been implicated in enhancing tumor progression. The insulin-like growth factor-II gene lies within the 11p15.5 chromosomal locus, which contains a number of other imprinted genes, and EGR1 expression was found to induce at least two other genes in this locus (IPL, p57(KIP2)). Based on our results, coupling adenoviral overexpression with microarray and quantitative reverse transcription-polymerase chain reaction analyses could be a versatile strategy for identifying target genes of transactivators.

Tissue factor expression and angiogenesis in human prostate carcinoma.

In tumors, the switch to the angiogenic phenotype is thought to be controlled by a balance of positive and negative angiogenic factors. Tissue factor (TF) produced by tumor cells has been implicated in the regulation of this "angiogenic switch" through its ability to concurrently induce the expression of angiogenic molecules such as vascular endothelial cell growth factor (VEGF), while inhibiting the expression of anti-angiogenic molecules such as thrombospondin 2. We have examined TF expression and its relationship to angiogenesis and tumor progression in human prostate carcinomas. Most of the prostate carcinoma specimens examined (73%; n = 67) express high levels of TF. Immunohistochemical analysis localized TF expression to the epithelial cells of malignant glands. TF expression was significantly correlated with tumor angiogenesis as measured by the microvessel density (MVD). In addition, TF expression was correlated with the preoperative PSA level, a strong predictor of recurrence in prostate carcinomas. Our findings show that TF expression by the malignant glands in prostate cancer is common and suggest a role for this molecule in regulating prostate cancer progression and angiogenesis.

High mobility group I/Y protein functions as a specific cofactor for Oct-2A: mapping of interaction domains.

The octamer motif (ATTTGCAT) present in several eukaryotic promoters and enhancers is now known to influence the transcription of several genes by interacting with members of a broad family of homeodomain proteins. The promoter of the human class II MHC gene HLA-DRA contains a conserved octamer element that can bind (among other proteins) the transcription factor Oct-2A and the high mobility group proteins (HMG) I/Y. We have previously determined that HMG I(Y) and Oct-2A cooperatively activate HLA-DRA gene expression, most likely due to the ability of HMG I(Y) to selectively recruit Oct-2A to the octamer motif. In this report, we present results of our investigations of the mechanisms of cooperative transactivation of HLA-DRA transcription by Oct-2A and HMG I(Y). We show that both the amino- and the carboxy-terminal domains of Oct-2A are required for HLA-DRA transactivation. Experiments using domain-swap chimeras of the Oct-1 and Oct-2A polypeptides indicate that cooperative activation of the DRA gene by HMG I(Y) and Oct-2A requires the carboxy-terminal domain (CTD) of Oct-2A. However, HMG I(Y) physically interacts with the conserved POU domains of both Oct-1 and Oct-2A. We therefore postulate that the nature of the CTD attached to the POU homeodomain influences the outcome of interaction with HMG I(Y). These studies support the view that HMG I(Y) is an important cofactor for HLA-DRA gene activation by Oct-2A and provide insights into its mechanism of action.

Inhibition of NF-AT-dependent transcription by NF-kappa B: implications for differential gene expression in T helper cell subsets.

Activation of individual CD4+ T cells results in differential lymphokine expression: interleukin 2 (IL-2) is preferentially produced by T helper type 1 (TH1) cells, which are involved in cell-mediated immune responses, whereas IL-4 is synthesized by TH2 cells, which are essential for humoral immunity. The Ca(2+)-dependent factor NF-ATp plays a key role in the inducible transcription of both these lymphokine genes. However, while IL2 expression requires the contribution of Ca(2+)- and protein kinase C-dependent signals, we report that activation of human IL4 transcription through the Ca(2+)-dependent pathway is diminished by protein kinase C stimulation in Jurkat T cells. This phenomenon is due to mutually exclusive binding of NF-ATp and NF-kappa B to the P sequence, an element located 69 bp upstream of the IL4 transcription initiation site. Human IL4 promoter-mediated transcription is downregulated in Jurkat cells stimulated with the NF-kappa B-activating cytokine tumor necrosis factor alpha and suppressed in RelA-overexpressing cells. In contrast, protein kinase C stimulation or RelA overexpression does not affect the activity of a human IL4 promoter containing a mouse P sequence, which is a higher-affinity site for NF-ATp and a lower-affinity site for RelA. Thus, competition between two general transcriptional activators, RelA and NF-ATp, mediates the inhibitory effect of protein kinase C stimulation on IL4 expression and may contribute to differential gene expression in TH cells.