First-in-human Evaluation of a Prostate-specific Membrane Antigen-targeted Near-infrared Fluorescent Small Molecule for Fluorescence-based Identification of Prostate Cancer in Patients with High-risk Prostate Cancer Undergoing Robotic-assisted Prostatectomy.

BACKGROUND: Men with high-risk prostate cancer undergoing surgery likely recur due to failure to completely excise regional and/or local disease. OBJECTIVE: The first-in-human evaluation of safety, pharmacokinetics, and exploratory efficacy of IS-002, a novel near-infrared prostate-specific membrane antigen (PSMA)-targeted fluorescence imaging agent, designed for intraoperative prostate cancer visualization. DESIGN, SETTING, AND PARTICIPANTS: A phase 1, single-center, dose-escalation study was conducted in 24 men with high-risk prostate cancer scheduled for robotic-assisted radical prostatectomy with (extended) pelvic lymph node dissection using the da Vinci surgical system. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Adverse events (AEs), vital signs, complete blood count, complete metabolic panel, urinalysis, and electrocardiogram were assessed over a 14-d period and compared with baseline. The pharmacokinetic profile of IS-002 was determined. Diagnostic accuracy was assessed for exploratory efficacy. RESULTS AND LIMITATIONS: AEs predominantly included discoloration of urine (n = 22/24; expected, related, grade 1). There were no grade ≥2 AEs. IS-002 Cmax and area under the curve increased with increasing dose. Plasma concentrations declined rapidly in a biphasic manner, with the median terminal half-lives ranging from 5.0 to 7.6 h, independent of dose and renal function. At 25 µg/kg, the exploratory efficacy readouts for the negative and positive predictive values were, 97% and 45% for lymph nodes, and 100% and 80% for residual/locoregional disease detection, respectively. CONCLUSIONS: IS-002 is safe and well tolerated, and has the potential to enable intraoperative tumor detection that could not be identified using standard imaging. PATIENT SUMMARY: IS-002 is a new imaging agent that specifically targets the prostate-specific membrane antigen receptor. In this study, we tested IS-002 for the first time in men with high-risk prostate cancer undergoing surgery and found that IS-002 is safe, is cleared from the body quickly, and potentially allows identification of prostate cancer in areas that would not be identified by conventional white light imaging.

ERα is an RNA-binding protein sustaining tumor cell survival and drug resistance.

Estrogen receptor α (ERα) is a hormone receptor and key driver for over 70% of breast cancers that has been studied for decades as a transcription factor. Unexpectedly, we discover that ERα is a potent non-canonical RNA-binding protein. We show that ERα RNA binding function is uncoupled from its activity to bind DNA and critical for breast cancer progression. Employing genome-wide cross-linking immunoprecipitation (CLIP) sequencing and a functional CRISPRi screen, we find that ERα-associated mRNAs sustain cancer cell fitness and elicit cellular responses to stress. Mechanistically, ERα controls different steps of RNA metabolism. In particular, we demonstrate that ERα RNA binding mediates alternative splicing of XBP1 and translation of the eIF4G2 and MCL1 mRNAs, which facilitates survival upon stress conditions and sustains tamoxifen resistance of cancer cells. ERα is therefore a multifaceted RNA-binding protein, and this activity transforms our knowledge of post-transcriptional regulation underlying cancer development and drug response.

Translation control of the immune checkpoint in cancer and its therapeutic targeting.

Cancer cells develop mechanisms to escape immunosurveillance, among which modulating the expression of immune suppressive messenger RNAs is most well-documented. However, how this is molecularly achieved remains largely unresolved. Here, we develop an in vivo mouse model of liver cancer to study oncogene cooperation in immunosurveillance. We show that MYC overexpression (MYCTg) synergizes with KRASG12D to induce an aggressive liver tumor leading to metastasis formation and reduced mouse survival compared with KRASG12D alone. Genome-wide ribosomal footprinting of MYCTg;KRASG12 tumors compared with KRASG12D revealed potential alterations in translation of mRNAs, including programmed-death-ligand 1 (PD-L1). Further analysis revealed that PD-L1 translation is repressed in KRASG12D tumors by functional, non-canonical upstream open reading frames in its 5' untranslated region, which is bypassed in MYCTg;KRASG12D tumors to evade immune attack. We show that this mechanism of PD-L1 translational upregulation was effectively targeted by a potent, clinical compound that inhibits eIF4E phosphorylation, eFT508, which reverses the aggressive and metastatic characteristics of MYCTg;KRASG12D tumors. Together, these studies reveal how immune-checkpoint proteins are manipulated by distinct oncogenes at the level of mRNA translation, which can be exploited for new immunotherapies.

Comparing Prognostic Utility of a Single-marker Immunohistochemistry Approach with Commercial Gene Expression Profiling Following Radical Prostatectomy.

BACKGROUND: Despite the availability of numerous genomic predictors of prostate cancer (PCa) outcome, few comparative studies have been performed. OBJECTIVE: To compare the prognostic utility of previously validated immunohistochemical (IHC) markers with an expression-based cell-cycle progression (CCP) score. DESIGN, SETTING, AND PARTICIPANTS: We identified 424 men with localized PCa treated with radical prostatectomy (RP). IHC analysis was performed using a tissue microarray to examine the expression status of PTEN, Ki-67, and ERG compared with previously calculated CCP scores derived from 31 genes normalized to 15 housekeeper genes. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Associations of IHC status and CCP scores, adjusted for clinical and pathologic characteristics were performed using Cox regression and competing risks regression to examine risk of biochemical recurrence (BCR), and metastasis or PCa-specific mortality (PCSM). We compared models using concordance index (c-index) testing. INTERVENTION: RP. RESULTS AND LIMITATIONS: Median age at treatment was 59 yr, and patients were followed for a median of 114 mo after RP. By 10 yr after RP, 27% experienced BCR and 4% developed metastasis or PCSM. In a multivariable model adjusted for Cancer of the Prostate Risk Assessment score (CAPRA-S), CCP was associated with risks of recurrence (hazard ratio [HR] 1.51, 95% confidence interval [CI] 1.08-2.11) and metastasis/PCSM (HR 2.15, 95% CI 1.36-3.39). PTEN loss was not associated with recurrence but was associated with metastasis/PCSM (HR 5.26, 95% CI 2.57-10.7), adjusted for CAPRA-S. The c-index for models consisting of PTEN status and CAPRA-S was similar (0.80) for risk of metastasis/PCSM when compared with CCP and CAPRA-S (0.81). Integration of Ki-67 and ERG status did not improve the c-index relative to CAPRA-S and PTEN alone. CONCLUSIONS: PTEN status offered comparable discrimination of the risk of metastasis or death from PCa relative to a commercial RNA amplification-based CCP assay. Efforts are warranted to reduce the cost of PCa prognostic tools in order to expand access. PATIENT SUMMARY: We compared a commercial genomic signature and the expression status of single genes to predict outcomes in men with prostate cancer who were treated with surgical removal. When accounting for clinical information about the patient's cancer, the status of the PTEN gene alone matched a multigene panel to predict which patient's cancer would metastasize or lead to death from the disease.

Development of a stress response therapy targeting aggressive prostate cancer.

Oncogenic lesions up-regulate bioenergetically demanding cellular processes, such as protein synthesis, to drive cancer cell growth and continued proliferation. However, the hijacking of these key processes by oncogenic pathways imposes onerous cell stress that must be mitigated by adaptive responses for cell survival. The mechanism by which these adaptive responses are established, their functional consequences for tumor development, and their implications for therapeutic interventions remain largely unknown. Using murine and humanized models of prostate cancer (PCa), we show that one of the three branches of the unfolded protein response is selectively activated in advanced PCa. This adaptive response activates the phosphorylation of the eukaryotic initiation factor 2-α (P-eIF2α) to reset global protein synthesis to a level that fosters aggressive tumor development and is a marker of poor patient survival upon the acquisition of multiple oncogenic lesions. Using patient-derived xenograft models and an inhibitor of P-eIF2α activity, ISRIB, our data show that targeting this adaptive brake for protein synthesis selectively triggers cytotoxicity against aggressive metastatic PCa, a disease for which presently there is no cure.

miR-124 and Androgen Receptor Signaling Inhibitors Repress Prostate Cancer Growth by Downregulating Androgen Receptor Splice Variants, EZH2, and Src.

miR-124 targets the androgen receptor (AR) transcript, acting as a tumor suppressor to broadly limit the growth of prostate cancer. In this study, we unraveled the mechanisms through which miR-124 acts in this setting. miR-124 inhibited proliferation of prostate cancer cells in vitro and sensitized them to inhibitors of androgen receptor signaling. Notably, miR-124 could restore the apoptotic response of cells resistant to enzalutamide, a drug approved for the treatment of castration-resistant prostate cancer. We used xenograft models to examine the effects of miR-124 in vivo when complexed with polyethylenimine-derived nanoparticles. Intravenous delivery of miR-124 was sufficient to inhibit tumor growth and to increase tumor cell apoptosis in combination with enzalutamide. Mechanistic investigations revealed that miR-124 directly downregulated AR splice variants AR-V4 and V7 along with EZH2 and Src, oncogenic targets that have been reported to contribute to prostate cancer progression and treatment resistance. Taken together, our results offer a preclinical rationale to evaluate miR-124 for cancer treatment.

Oncomir miR-125b suppresses p14(ARF) to modulate p53-dependent and p53-independent apoptosis in prostate cancer.

MicroRNAs are a class of naturally occurring small non-coding RNAs that target protein-coding mRNAs at the post-transcriptional level and regulate complex patterns of gene expression. Our previous studies demonstrated that in human prostate cancer the miRNA miR-125b is highly expressed, leading to a negative regulation of some tumor suppressor genes. In this study, we further extend our studies by showing that miR-125b represses the protein product of the ink4a/ARF locus, p14(ARF), in two prostate cancer cell lines, LNCaP (wild type-p53) and 22Rv1 (both wild type and mutant p53), as well as in the PC-346C prostate cancer xenograft model that lentivirally overexpressed miR-125b. Our results highlight that miR-125b modulates the p53 network by hindering the down-regulation of Mdm2, thereby affecting p53 and its target genes p21 and Puma to a degree sufficient to inhibit apoptosis. Conversely, treatment of prostate cancer cells with an inhibitor of miR-125b (anti-miR-125b) resulted in increased expression of p14(ARF), decreased level of Mdm2, and induction of apoptosis. In addition, overexpression of miR-125b in p53-deficient PC3 cells induced down-regulation of p14(ARF), which leads to increased cell proliferation through a p53-independent manner. Thus, we conclude that miR-125b acts as an oncogene which regulates p14(ARF)/Mdm2 signaling, stimulating proliferation of prostate cancer cells through a p53-dependent or p53-independent function. This reinforces our belief that miR-125b has potential as a therapeutic target for the management of patients with metastatic prostate cancer.

Dual EGFR/HER2 inhibition sensitizes prostate cancer cells to androgen withdrawal by suppressing ErbB3.

PURPOSE: Patients with recurrent prostate cancer are commonly treated with androgen withdrawal therapy (AWT); however, almost all patients eventually progress to castration resistant prostate cancer (CRPC), indicating failure of AWT to eliminate androgen-sensitive prostate cancer. The overall goal of these studies is to determine whether dual inhibition of the receptor tyrosine kinases epidermal growth factor receptor (EGFR) and HER2 would prolong the effectiveness of this treatment in prostate cancer. EXPERIMENTAL DESIGN: We used androgen-dependent LNCaP cells and its CRPC sublines LNCaP-AI and C4-2. Additional data were collected in pRNS-1-1 cells stably expressing a mutant androgen receptor (AR-T877A), and in nude mice harboring CWR22 tumors. Studies utilized EGFR inhibitors erlotinib and AG1478, and HER2 inhibitors trastuzumab and AG879. RESULTS: Dual EGFR/HER2 inhibition induced apoptosis selectively in androgen-sensitive prostate cancer cells undergoing AWT, but not in the presence of androgens, or in CRPC cells. We show that AWT alone failed to induce significant apoptosis in androgen-dependent cells, due to AWT-induced increase in HER2 and ErbB3, which promoted survival by increasing Akt phosphorylation. AWT-induced ErbB3 stabilized the AR and stimulated PSA, while it was inactivated only by inhibition of both its dimerization partners EGFR and HER2 (prostate cancer cells do not express ErbB4); but not the inhibition of any one receptor alone, explaining the success of dual EGFR/HER2 inhibition in sensitizing androgen-dependent cells to AWT. The effectiveness of the inhibitors in suppressing growth correlated with its ability to prevent Akt phosphorylation. CONCLUSION: These studies indicate that dual EGFR/HER2 inhibition, administered together with AWT, sensitize prostate cancer cells to apoptosis during AWT.

Influence of short polyglutamine tracts and p160 coactivators on the transactivation of the androgen receptor.

The androgen receptor (AR) acting as a transcription factor plays a pivotal role in the occurrence and progression of prostate cancer (CaP). Several AR-related factors or modulators have been reported to influence AR activity. Whether and how these factors cooperatively modulate the AR activity has not been well defined. In the present study, the combined effect of p160 coactivators, short CAG length (encoding a short polyQ tract), and AR mutations on AR transactivation in a yeast system was evaluated. It was found that the short polyQ tract can upregulate the transactivation of the wild-type (WT) AR and partial-function (PF) AR mutants in response to a physiological level (10(-9) M) of dihydrotestosterone. Addition of a p160 coactivator (SRC-1 or TIF2) to the above systems resulted in a significant increase in the ligand-stimulated transactivation. Although the androgen antagonist bicalutamide could suppress the activity of androgen-activated WT or PF ARs, it was unable to do so for gain-of-function AR mutants. A combination of the short polyQ tract and coactivator TIF2 acted cooperatively on the WT AR and PF AR mutants to enhance their transactivation in response to either a low level of dihydrotestosterone (10(-10) M) or adrenal dehydroepiandrosterone. Taken together, this finding suggests that the modulated AR activity may involve early in the carcinogenesis of CaP. Additionally, these data support the concept that a given CaP in which the AR activity is modulated by multiple AR modulators may progress more readily to castrate resistance.

miR-125b promotes growth of prostate cancer xenograft tumor through targeting pro-apoptotic genes.

BACKGROUND: Increasing evidence demonstrates that aberrantly regulated microRNAs (miRNAs) contribute to the initiation and progression of human cancer. We previously have demonstrated that miR-125b stimulated the growth of prostate cancer (CaP) cells. In this study, we further determined the influence of miR-125b on the pathogenesis of CaP. METHODS: To evaluate the effect of miR-125b on xenograft tumor growth, male athymic mice were subcutaneously injected with PC-346C-miR-125b cells that stably overexpressed miR-125b. Potential direct target transcripts of miR-125b were identified using a bioinformatics approach and three miR-125b targeted molecules were confirmed by means of biochemical analyses. RESULTS: Enforced expression of miR-125b promoted tumor growth in both intact and castrated male nude mice. In an effort to define the molecular mechanism(s) mediating its tumor growth properties, we found that miR-125b directly targets eight transcripts, including three key pro-apoptotic genes: p53, Puma, and Bak1. Increasing the abundance of miR-125b resulted in a dramatic decrease in the levels of these three proteins in CaP cells. A direct repressive effect on each of these was supported by the ability of miR-125b to significantly reduce the activity of luciferase reporters containing their 3'-untranslated regions of each gene encompassing the miR-125b-binding sites. Additionally, we found that repression of miR-125b activity was able to sensitize CaP cells to different therapeutic interventions. CONCLUSION: Data obtained in this study demonstrate that miR-125b promotes growth of prostatic xenograft tumors by down-regulating three key pro-apoptotic genes. This suggests that miR-125b is oncogenic and makes it an attractive therapeutic target in CaP.

Prolonged androgen receptor loading onto chromatin and the efficient recruitment of p160 coactivators contribute to androgen-independent growth of prostate cancer cells.

BACKGROUND: Growth of most ablation-resistant prostate cancers (CaPs) is dependent on androgen receptor (AR) activity in chromatin, but cancer cells in these tumors have acquired altered AR activation. It is unclear how the aberrantly activated AR loads onto regulatory regions of AR-targeted genes. The purpose of this study was to assess the AR chromatin loading in an androgen-depleted environment. METHODS: The expression of PSA in androgen-resistant CaP cells was determined using RT-PCR and Western blot analysis. In order to investigate the binding of the AR to the PSA gene regulatory regions, chromatin immunoprecipitation (ChIP) was performed in the androgen-independent cds2 cell line in the presence or absence of androgens. In addition, we examined the involvement of p160 coactivators in the chromatin loading of the AR. RESULTS: It was found that constitutive activation of PSA expression was the result of sustained occupancy by the AR at the regulatory region of this gene. This stable AR loading was not blocked by the AR antagonist bicalutamide. Furthermore, androgen-resistant CaP cells highly expressed both AR and the p160 coactivators and the AR was able to recruit TIF2. Downregulation of TIF2 using short hairpin RNA disrupted the AR loading to the PSA enhancer and subsequently inhibited AR activity. CONCLUSION: Prolonged AR localization to the regulatory regions of AR targeted genes and the recruitment of p160 coactivators are a potential mechanism leading to androgen-independent activation of the AR. Disruption of AR chromatin loading could therefore become an important therapeutic target for this disease.

An androgen-regulated miRNA suppresses Bak1 expression and induces androgen-independent growth of prostate cancer cells.

Although prostate cancer (CaP) is the most frequently diagnosed malignant tumor and the second leading cause of cancer deaths in American men, the mechanisms explaining the development and progression of CaP remain largely unknown. Recent studies have shown that some aberrantly expressed microRNAs (miRNAs) are involved in tumorigenesis. Although aberrant expression of certain miRNAs has been discovered in CaP, their function in this disease has not yet been defined. In this study, we found differential expression of miR-125b in androgen-dependent and independent CaP cells, as well as in benign and malignant prostate tissues. Furthermore, androgen signaling was able to up-regulate the expression of miR-125b. In addition, transfection of synthetic miR-125b stimulated androgen-independent growth of CaP cells and down-regulated the expression of Bak1. Our results suggest that miR-125b acts as an oncogene, contributing to the pathogenesis of CaP.

The oncogenic potential of a prostate cancer-derived androgen receptor mutant.

BACKGROUND: The role of androgen receptor (AR) mutations in the initiation of prostate cancer (CaP) remains unclear. The purpose of this study was to assess the influence of an AR mutation on prostate tumorigenesis and to determine the resulting molecular alterations. METHODS: Wild-type AR (AR(WT)) or the CaP-derived K580R AR (AR(K580R)) mutant was stably transfected into SV40-immortalized human prostate epithelial pRNS-1-1 cells that lack AR expression and fail to grow in nude mice. The ability of these AR-transfected cell lines to form tumor was investigated in vitro and in vivo. Additionally, gene expression profiling of these cell lines was performed. RESULTS: Compared with the AR(WT), the AR(K580R) induced greater than sixfold increase in colony formation in soft agar. In vivo studies confirmed that the AR(K580R)-transfected pRNS-1-1 cells were able to form tumors in nude mice. Using a combination of microarray and RT-PCR, 29 differentially expressed genes were identified in AR(K580R) cells. It was found that silencing the expression of placental alkaline phosphatase (ALPP) that was upregulated in AR(K580R) cells resulted in significant inhibition of cell growth. Furthermore, the AR(K580R)-transfected pRNS-1-1 cells expressed markedly increased p-Akt and p-p70 S6K. CONCLUSION: The AR(K580R) mutation promoted the malignant transformation of prostate epithelial cells. This was associated with upregulation of ALPP and subsequent activation of the Akt signaling pathway.

GCP-mediated growth inhibition and apoptosis of prostate cancer cells via androgen receptor-dependent and -independent mechanisms.

BACKGROUND: Genistein combined polysaccharide (GCP) is a nutritional supplement that can inhibit prostate cancer growth experimentally and clinically. It is composed predominantly of the isoflavones genistein, daidzein, and glycitein, which have anti-cancer properties. Although genistein is well studied, the properties of GCP are not well defined. The goal of this work was to better characterize the signaling pathways impacted by GCP in an effort to optimize its efficacy. METHODS: Cell growth and apoptosis were evaluated by MTS proliferation, caspase-based assays, and flow cytometry. Modulation of androgen receptor (AR) levels and activation status of signaling molecules were monitored by immunoblot analysis. AR function was measured by evaluating prostate-specific antigen (PSA) message and protein levels and by reporter assays. RESULTS: GCP inhibited proliferation of androgen-dependent LNCaP and androgen-independent LNCaP-p53(GOF) and 22Rv1 cell lines in a dose-dependent manner and cells were more responsive in the presence of androgen. GCP markedly suppressed mTOR-p70S6K signaling while Akt and p53 were only modestly modulated. GCP significantly attenuated androgen signaling as evidenced by diminished AR protein levels and a consequent reduction in transcriptional activity and PSA expression. AR expression was enhanced by de-repression of translation with inhibitors of PI3K-Akt-mTOR signaling and by inhibition of proteasome-dependent degradation. Neither inhibitor could counteract GCP-mediated AR downregulation, suggesting the involvement of a mechanism(s) independent of these pathways. CONCLUSIONS: Our results suggest that GCP mediates growth inhibition and apoptosis through multiple mechanisms including (1) molecular mimicry of androgen ablation (via AR downregulation) and (2) by providing an AR-independent, pro-apoptotic signal (mTOR inhibition).

The MsPRP2 promoter enables strong heterologous gene expression in a root-specific manner and is enhanced by overexpression of Alfin 1.

Promoter specificity and efficiency of utilization are essential for endogenous and transgene expression. Selective root expression remains to be defined in terms of both promoter elements and transcription factors that provide high levels of ubiquitous expression. We characterized expression from the MsPRP2 promoter with the green fluorescent protein (GFP) reporter transgene in alfalfa (Medicago sativa) and found that a promoter fragment (+1 to -652 bp) retained the root and callus specificity of the endogenous MsPRP2 gene and hence this promoter fragment contains elements necessary for root-specific expression. The strong ubiquitous expression obtained from this promoter was comparable to that of the CaMV 35S promoter in roots and was enhanced by transgenic overexpression of Alfin 1, a root- and callus-specific transcription factor in alfalfa. No transgenic expression was obtained in leaves with this promoter in the presence or absence of Alfin 1. The increased expression of GFP in alfalfa containing the Alfin 1 transgene confirms the function of Alfin 1 binding sites in the MsPRP2 promoter fragment and also indicates that Alfin 1 concentrations are limiting for maximal expression in calli and roots. These findings characterize the MsPRP2 promoter as a novel root- and callus-specific promoter of plant origin that can be used as an effective tool for strong root-directed gene expression. In addition, we have demonstrated that the signal sequence of MsPRP2 can be used for efficient secretion of transgene products from callus and roots.

Re-examining alveolate evolution using multiple protein molecular phylogenies.

Alveolates are a diverse group of protists that includes three major lineages: ciliates, apicomplexa, and dinoflagellates. Among these three, it is thought that the apicomplexa and dinoflagellates are more closely related to one another than to ciliates. However, this conclusion is based almost entirely on results from ribosomal RNA phylogeny because very few morphological characters address this issue and scant molecular data are available from dinoflagellates. To better examine the relationships between the three major alveolate groups, we have sequenced six genes from the non-photosynthetic dinoflagellate, Crypthecodinium cohnii: actin, beta-tubulin, hsp70, BiP, hsp90, and mitochondrial hsp10. Beta-tubulin, hsp70, BiP, and hsp90 were found to be useful for intra-alveolate phylogeny, and trees were inferred from these genes individually and in combination. Trees inferred from individual genes generally supported the apicomplexa-dinoflagellate grouping, as did a combined analysis of all four genes. However, it was also found that the outgroup had a significant effect on the topology within alveolates when using certain methods of phylogenetic reconstruction, and an alternative topology clustering dinoflagellates and ciliates could not be rejected by the combined data. Altogether, these results support the sisterhood of apicomplexa and dinoflagellates, but point out that the relationship is not as strong as is often assumed.