Pleckstrin-2 Mediates the Activation of AKT in Prostate Cancer and Is Repressed by Androgen Receptor.

Phosphoinositide 3-kinase (PI3K)-AKT and androgen receptor (AR) pathways are commonly activated in prostate cancers. Their reciprocal regulation makes advanced prostate cancers difficult to treat. The current study shows that pleckstrin-2 (PLEK2), a proto-oncoprotein involved in the activation and stabilization of AKT, connects these two pathways. Genetic evidence provided herein suggests that Plek2 deficiency largely reverted tumorigenesis in Pten prostate-specific knockout mice and that overexpression of PLEK2 promoted the proliferation and colony formation of prostate cancer cells in vitro. In addition, PLEK2 was negatively regulated by AR, AR transcriptionally repressed PLEK2 through binding to the PLEK2 promoter region, and overexpression of AR reduced PLEK2 expression, which inactivated AKT. Conversely, knockdown of AR in prostate cancer cells increased PLEK2 expression and activated the AKT pathway. This reciprocal inhibitory loop can be pharmacologically targeted using the PLEK2 inhibitor. PLEK2 inhibitor dose-dependently inhibited prostate cancer cell proliferation with the inactivation of AKT. Overall, the current study uncovered a crucial role of PLEK2 in prostate cancer proliferation and provided the rationale for targeting PLEK2 to treat prostate cancers.

Hematological, clinical, immunophenotypic characterization, and treatment outcomes of prognostically significant genetic subtypes of B-lineage acute lymphoblastic leukemia: A report of 1021 patients from India.

BACKGROUND: The published literature on hematological, clinical, flowcytometric-immunophenotyping, and minimal residual disease outcomes of the prognostically important genetic subtypes of acute lymphoblastic leukemia (ALL) is scarce from low-income countries. For newer classifications such as BCR::ABL1-like ALLs, the scarcity of patient-level data is even more pronounced. METHODS: The authors performed comprehensive detection of recurrent gene fusions and BCR::ABL1-like ALL cases followed by immunophenotypic profiling and obtained clinical outcome parameters for a large cohort (n = 1021) of patients from India. This cohort included a significant number of patients with BCR::ABL1-like ALL subtype and other genetic subtypes of ALL. RESULTS: Patients with BCR::ABL1-positive and BCR::ABL1-like ALL were significantly older, had male preponderance, and expressed a higher white blood cell count than BCR::ABL1-negative cases (p < .05). Logistic regression modeling of B-lineage-ALL (B-ALL) subtypes revealed that cluster of differentiation (CD)36 is a strong statistically significant predictive marker of BCR::ABL1-like ALL (p < .05). Furthermore, patients with BCR::ABL1-like ALLs show a significantly higher frequency of CD36 expression compared to BCR::ABL1-negative ALLs (p < .05). In terms of clinical symptoms, lymphadenopathy is a strong statistically significant predictive marker in BCR::ABL1-like ALLs compared to BCR::ABL1-negative ALL cases (p < .05). In terms of treatment outcomes, minimal residual disease (MRD) positivity in BCR::ABL1-positive ALL cases were statistically significant (p < .05), and BCR::ABL1-like ALL cases had high MRD-positivity as compared to BCR::ABL1-negative ALL cases but did not show statistical significance. CONCLUSIONS: The findings evince the use of novel therapies and personalized treatment regimens to improve the overall survival of the newer incorporated entities in B-ALLs. This is the first report characterizing the hematological, clinical, flowcytometric-immunophenotyping, and minimal residual disease outcomes of the prognostically significant subtypes of ALLs in patients from India. PLAIN LANGUAGE SUMMARY: Characterizing the hematological, clinical, flowcytometric-immunophenotyping, and minimal residual disease outcomes of the prognostically significant subtypes (n = 1021) of acute lymphoblastic leukemia (ALLs) in patients from India. We have made two independent logistic regression models of cluster of differentiation (CD) markers and clinical symptoms to differentiate prognostically significant subtypes of ALLs. Logistic regression analysis of CD markers revealed CD36 as a strong predictor in BCR::ABL1-like ALL cases compared to BCR::ABL1-negative ALL cases. Logistic regression analysis of clinical symptoms revealed lymphadenopathy significantly predicts BCR::ABL1-like ALLs (p < .05). In terms of treatment outcomes, BCR::ABL1-positive ALL had statistically significant minimal residual disease (MRD) (p < .05), and BCR::ABL1-like ALL cases had high MRD-positivity but did not show statistical significance as compared to BCR::ABL1-negative ALLs.

Advanced glycation end-products (AGEs) are lower in prostate tumor tissue and inversely related to proportion of West African ancestry.

BACKGROUND: The metabolism of normal prostate relies on glycolysis, with prostate cancer having reduced glycolysis and increased aerobic metabolism. Advanced glycation end products (AGEs) accumulate in tissues as a result of age and glycolytic rate. Differential AGE levels were recently observed in prostate cancer tissues. Herein we sought to quantify AGEs in benign and cancer prostate tissue in a diverse cohort of patients. METHODS: Levels of the AGE Nε-(carboxylethyl)lysine (CML) were quantified by immunohistochemistry (IHC) in a tissue microarray which consisted of 3 cores from tumor and 2 cores from benign areas from 118 patients (87 African American and 31 European American). Ancestry informative markers for African Ancestry were available for 79 patients. Epithelial and stromal areas were quantified separately using an E-cadherin mask. CML levels were compared with clinical grade group and ancestry by mixed linear effect models. Age, prostate-specific antigen (PSA) levels, body mass index (BMI), and hemoglobin A1C were included as covariates. RESULTS: CML levels were lower in areas of the tumor, for both epithelium and surrounding stroma, compared with benign, but did not significantly change with tumor grade group. Age, PSA levels, BMI, and hemoglobin A1C did not associate with CML levels. CML levels were inversely associated with the percentage of African Ancestry in all tissues. CONCLUSIONS: The low CML levels in cancer may reflect the reduced glycolytic state of the tissue. The inverse relationship between African Ancestry and CML levels in both benign and cancer areas suggests a state of reduced glycolysis. It is yet to be determined whether altered glycolysis and CML levels are bystanders or drivers of carcinogenesis.

Association between inflammatory bowel disease and prostate cancer: A large-scale, prospective, population-based study.

Inflammatory bowel disease (IBD) is an established risk factor for colorectal cancer. Recent reports suggesting IBD is also a risk factor for prostate cancer (PC) require further investigation. We studied 218 084 men in the population-based UK Biobank cohort, aged 40 to 69 at study entry between 2006 and 2010, with follow-up through mid-2015. We assessed the association between IBD and subsequent PC using multivariable Cox regression analyses, adjusting for age at assessment, ethnic group, UK region, smoking status, alcohol drinking frequency, body mass index, Townsend Deprivation Index, family history of PC and previous prostate-specific antigen testing. Mean age at study entry was 56 years, 94% of the men were white, and 1.1% (n = 2311) had a diagnosis of IBD. After a median follow-up of 78 months, men with IBD had an increased risk of PC (adjusted hazard ratio [aHR] = 1.31, 95% confidence interval [CI] = 1.03-1.67, P = .029). The association with PC was only among men with the ulcerative colitis (UC; aHR = 1.47, 95% CI = 1.11-1.95, P = .0070), and not Crohn's disease (aHR 1.06, 95% CI = 0.63-1.80, P = .82). Results are limited by lack of data on frequency of health care interactions. In a large-scale, prospective cohort study, we detected an association between IBD, and UC specifically, with incident PC diagnosis.

RAMP1 is a direct NKX3.1 target gene up-regulated in prostate cancer that promotes tumorigenesis.

The homeodomain-containing transcription factor, NKX3.1, plays an important role in the suppression of prostate tumorigenesis. Herein, we identify the receptor activity-modifying protein 1 (RAMP1) as a direct NKX3.1 target gene through analysis of chromatin immunoprecipitation coupled to massively parallel sequencing and gene expression data. RAMP1 is a coreceptor for certain G-protein-coupled receptors, such as the calcitonin gene-related peptide receptor, to the plasma membrane. We found that RAMP1 expression is specifically elevated in human prostate cancer relative to other tumor types. Furthermore, RAMP1 mRNA and protein levels are significantly higher in human prostate cancer compared with benign glands. We identified multiple NKX3.1 binding sites in the RAMP1 locus in human prostate cancer cells and in the normal mouse prostate. Analyses of Nkx3.1 knockout mice and human prostate cancer cell lines indicate that NKX3.1 represses RAMP1 expression. Knockdown of RAMP1 by shRNA decreased prostate cancer cell proliferation and tumorigenicity in vitro and in vivo. By using gene expression profiling and pathway analyses, we identified several cancer-related pathways that are significantly altered in RAMP1 knockdown cells, including the mitogen-activated protein kinase signaling pathway. Further experiments confirmed a reduction in MAP2KI (MEK1) expression and phosphorylated-extracellular signal-regulated kinase 1/2 levels in RAMP1 knockdown cells. These data provide novel insights into the role of RAMP1 in promoting prostate tumorigenesis and support the potential of RAMP1 as a novel biomarker and possible therapeutic target in prostate cancer.

Asparagine Dependency Is a Targetable Metabolic Vulnerability in TP53-Altered Castration-Resistant Prostate Cancer.

TP53 tumor suppressor is frequently altered in lethal, castration-resistant prostate cancer (CRPC). However, to date there are no effective treatments that specifically target TP53 alterations. Using transcriptomic and metabolomic analyses, we have shown here that TP53-altered prostate cancer exhibits an increased dependency on asparagine (Asn) and overexpresses Asn synthetase (ASNS), the enzyme catalyzing the synthesis of Asn. Mechanistically, the loss or mutation of TP53 transcriptionally activated ASNS expression, directly and via mTORC1-mediated ATF4 induction, driving de novo Asn biosynthesis to support CRPC growth. TP53-altered CRPC cells were sensitive to Asn restriction by knockdown of ASNS or L-asparaginase treatment to deplete the intracellular and extracellular sources of Asn, respectively, and cell viability was rescued by Asn addition. Notably, pharmacological inhibition of intracellular Asn biosynthesis using a glutaminase inhibitor and depletion of extracellular Asn with L-asparaginase significantly reduced Asn production and effectively impaired CRPC growth. This study highlights the significance of ASNS-mediated metabolic adaptation as a synthetic vulnerability in CRPC with TP53 alterations, providing a rationale for targeting Asn production to treat these lethal prostate cancers. Significance: TP53-mutated castration-resistant prostate cancer is dependent on asparagine biosynthesis due to upregulation of ASNS and can be therapeutically targeted by approaches that deplete intracellular and extracellular asparagine.

Mechanism-centric regulatory network identifies NME2 and MYC programs as markers of Enzalutamide resistance in CRPC.

Heterogeneous response to Enzalutamide, a second-generation androgen receptor signaling inhibitor, is a central problem in castration-resistant prostate cancer (CRPC) management. Genome-wide systems investigation of mechanisms that govern Enzalutamide resistance promise to elucidate markers of heterogeneous treatment response and salvage therapies for CRPC patients. Focusing on the de novo role of MYC as a marker of Enzalutamide resistance, here we reconstruct a CRPC-specific mechanism-centric regulatory network, connecting molecular pathways with their upstream transcriptional regulatory programs. Mining this network with signatures of Enzalutamide response identifies NME2 as an upstream regulatory partner of MYC in CRPC and demonstrates that NME2-MYC increased activities can predict patients at risk of resistance to Enzalutamide, independent of co-variates. Furthermore, our experimental investigations demonstrate that targeting MYC and its partner NME2 is beneficial in Enzalutamide-resistant conditions and could provide an effective strategy for patients at risk of Enzalutamide resistance and/or for patients who failed Enzalutamide treatment.

Tumorigenic polyploid cells contain elevated ROS and ARE selectively targeted by antioxidant treatment.

Polyploidy has been linked to tumorigenicity mainly due to the chromosomal aberrations. Elevated reactive oxygen species (ROS) generation, on the other hand, has also been associated with oncogenic transformation in most cancer cells. However, a possible link between ploidy and ROS is largely unexplored. Here we have examined the role of ROS in the tumorigenicity of polyploid cells. We show that polyploid prostate and mammary epithelial cells contain higher levels of ROS due to their higher mitochondrial contents. ROS levels and mitochondrial mass are also higher in dihydrocytochalasin B (DCB)-induced polyploid cells, suggesting that higher levels of ROS observed in polyploid cell can occur due to cytokinesis failure. Interestingly, polyploid cells were more sensitive to the inhibitory effect of the antioxidant, N-Acetyl-L-cysteine (NAC), than control diploid cells. Treatment of polyploid/diploid cells with NAC led to the selective elimination of polyploid cells over time and abrogated the tumorigenicity of polyploid cells. This effect was partially mediated via the Akt signaling pathway. We next explored a possible role for ROS in promoting chromosomal instability by analyzing the effects of ROS on the mitotic stage of the cell cycle. Enhancing ROS levels by treating cells with hydrogen peroxide delayed not only entry into and but also exit from mitosis. Furthermore, increasing ROS levels significantly increased taxol resistance. Our results indicated that increased ROS in polyploid cells can contribute to tumorigenicity and highlight the therapeutic potential of antioxidants by selectively targeting the tumorigenic polyploid cells and by reversing taxol resistance.

Haploinsufficiency at the Nkx3.1 locus. A paradigm for stochastic, dosage-sensitive gene regulation during tumor initiation.

Tumorigenesis requires sequential accumulation of multiple genetic lesions. In the prostate, tumor initiation is often linked to loss of heterozygosity at the Nkx3.1 locus. In mice, loss of even one Nkx3.1 allele causes prostatic epithelial hyperplasia and eventual prostatic intraepithelial neoplasia (PIN) formation. Here we demonstrate that Nkx3.1 allelic loss extends the proliferative stage of regenerating luminal cells, leading to epithelial hyperplasia. Microarray analysis identified Nkx3.1 target genes, many of which show exquisite dosage sensitivity. The number of Nkx3.1 alleles determines the relative probabilities of stochastic activation or inactivation of a given target gene. Thus, loss of a single Nkx3.1 allele likely results in hyperplasia and PIN by increasing the probability of completely inactivating select Nkx3.1-regulated pathways within a subset of affected cells.

Interactions between cells with distinct mutations in c-MYC and Pten in prostate cancer.

In human somatic tumorigenesis, mutations are thought to arise sporadically in individual cells surrounded by unaffected cells. This contrasts with most current transgenic models where mutations are induced synchronously in entire cell populations. Here we have modeled sporadic oncogene activation using a transgenic mouse in which c-MYC is focally activated in prostate luminal epithelial cells. Focal c-MYC expression resulted in mild pathology, but prostate-specific deletion of a single allele of the Pten tumor suppressor gene cooperated with c-MYC to induce high grade prostatic intraepithelial neoplasia (HGPIN)/cancer lesions. These lesions were in all cases associated with loss of Pten protein expression from the wild type allele. In the prostates of mice with concurrent homozygous deletion of Pten and focal c-MYC activation, double mutant (i.e. c-MYC+;Pten-null) cells were of higher grade and proliferated faster than single mutant (Pten-null) cells within the same glands. Consequently, double mutant cells outcompeted single mutant cells despite the presence of increased rates of apoptosis in the former. The p53 pathway was activated in Pten-deficient prostate cells and tissues, but c-MYC expression shifted the p53 response from senescence to apoptosis by repressing the p53 target gene p21(Cip1). We conclude that c-MYC overexpression and Pten deficiency cooperate to promote prostate tumorigenesis, but a p53-dependent apoptotic response may present a barrier to further progression. Our results highlight the utility of inducing mutations focally to model the competitive interactions between cell populations with distinct genetic alterations during tumorigenesis.

A Genome-Wide CRISPR Activation Screen Identifies PRRX2 as a Regulator of Enzalutamide Resistance in Prostate Cancer.

Androgen receptor (AR) pathway inhibitors are the mainstay treatment for advanced prostate cancer, but resistance to therapy is common. Here, we used a CRISPR activation screen in metastatic castration-sensitive prostate cancer cells to identify genes that promote resistance to AR inhibitors. Activation of the TGFß target gene paired-related homeobox2 (PRRX2) promoted enzalutamide resistance. PRRX2 expression was the highest in double-negative prostate cancer (DNPC), which lack AR signaling and neuroendocrine differentiation, and a PRRX2-related gene signature identified a subset of patients with DNPC with reduced overall survival. PRRX2-expressing cells showed alterations in the CDK4/6/Rb/E2F and BCL2 pathways. Accordingly, treatment with CDK4/6 and BCL2 inhibitors sensitized PRRX2-expressing, castration-resistant tumors to enzalutamide. Overall, PRRX2 was identified as a driver of enzalutamide resistance. The PRRX2 signature merits investigation as a biomarker of enzalutamide resistance in prostate cancer that could be reversed with CDK4/6 and BCL2 inhibitors. SIGNIFICANCE: PRRX2 mediates enzalutamide resistance via activation of the E2F and BCL2 pathways, which can be targeted with CDK4/6 and BCL2 inhibitors to reverse resistance.

A MYC inhibitor selectively alters the MYC and MAX cistromes and modulates the epigenomic landscape to regulate target gene expression.

MYC regulates multiple gene programs, raising questions about the potential selectivity and downstream transcriptional consequences of MYC inhibitors as cancer therapeutics. Here, we examined the effect of a small-molecule MYC inhibitor, MYCi975, on the MYC/MAX cistromes, epigenome, transcriptome, and tumorigenesis. Integrating these data revealed three major classes of MYCi975-modulated gene targets: type 1 (down-regulated), type 2 (up-regulated), and type 3 (unaltered). While cell cycle and signal transduction pathways were heavily targeted by MYCi, RNA biogenesis and core transcriptional pathway genes were spared. MYCi975 altered chromatin binding of MYC and the MYC network family proteins, and chromatin accessibility and H3K27 acetylation alterations revealed MYCi975 suppression of MYC-regulated lineage factors AR/ARv7, FOXA1, and FOXM1. Consequently, MYCi975 synergistically sensitized resistant prostate cancer cells to enzalutamide and estrogen receptor-positive breast cancer cells to 4-hydroxytamoxifen. Our results demonstrate that MYCi975 selectively inhibits MYC target gene expression and provide a mechanistic rationale for potential combination therapies.

Inflammatory bowel disease induces inflammatory and pre-neoplastic changes in the prostate.

BACKGROUND: Inflammatory bowel disease (IBD) has been implicated as a risk factor for prostate cancer, however, the mechanism of how IBD leads to prostate tumorigenesis is not known. Here, we investigated whether chronic intestinal inflammation leads to pro-inflammatory changes associated with tumorigenesis in the prostate. METHODS: Using clinical samples of men with IBD who underwent prostatectomy, we analyzed whether prostate tumors had differences in lymphocyte infiltrate compared to non-IBD controls. In a mouse model of chemically-induced intestinal inflammation, we investigated whether chronic intestinal inflammation could be transferred to the wild-type mouse prostate. In addition, mouse prostates were evaluated for activation of pro-oncogenic signaling and genomic instability. RESULTS: A higher proportion of men with IBD had T and B lymphocyte infiltration within prostate tumors. Mice with chronic colitis showed significant increases in prostatic CD45 + leukocyte infiltration and elevation of three pro-inflammatory cytokines-TIMP-1, CCL5, and CXCL1 and activation of AKT and NF-kB signaling pathways. Lastly, mice with chronic colitis had greater prostatic oxidative stress/DNA damage, and prostate epithelial cells had undergone cell cycle arrest. CONCLUSIONS: These data suggest chronic intestinal inflammation is associated with an inflammatory-rich, pro-tumorigenic prostatic phenotype which may explain how gut inflammation fosters prostate cancer development in men with IBD.

Turning Up the Heat on MYC: Progress in Small-Molecule Inhibitors.

MYC is a highly validated oncogenic transcription factor and cancer target. However, the disordered nature of this protein has made it a challenging target, with no clinical stage, direct small-molecule MYC inhibitors available. Recent work leveraging a large in silico chemical library and a rapid in vivo screen has expanded the chemotypes of direct small-molecule inhibitors (MYCi). Novel MYCi represent a class of improved MYC chemical probes that bind directly to MYC to inhibit its function and to promote its degradation by enhancing GSK3ß-mediated phosphorylation. One of these compounds, MYCi975, has shown remarkable tolerability and efficacy in vivo and is associated with a selective effect on MYC target gene expression. Additional effects of MYCi on the tumor immune microenvironment including immune cell infiltration and upregulation of PD-L1 expression provide a rationale for combining MYCi with anti-PD-1/PD-L1 therapy to enhance antitumor efficacy. Our strategy for developing MYCi demonstrates an efficient way to identify selective and well-tolerated MYC inhibitors. The new MYCi provide tools for probing MYC function and serve as starting points for the development of novel anti-MYC therapeutics.

Macrophages expedite cell proliferation of prostate intraepithelial neoplasia through their downstream target ERK.

The risk factors for prostate cancer include a high-fat diet and obesity, both of which are associated with an altered cell environment including increased inflammation. It has been shown that chronic inflammation due to a high-fat diet or bacterial infection has the potential to accelerate prostate cancer as well as its precursor, prostatic intraepithelial neoplasia (PIN), development. However, the underlying mechanism of how chronic inflammation promotes prostate cancer development, especially PIN, remains unclear. In this study, we showed that more macrophages were present in PIN areas as compared to the normal areas of human prostate. When co-culturing PIN cells with macrophages in 3D, more PIN cells had nuclear localized cyclin D1, indicating that macrophages enhanced PIN cell proliferation. We identified ICAM-1 and CCL2 as chemoattractants expressed by PIN cells to recruit macrophages. Furthermore, we discovered that macrophage-secreted cytokines including C5a, CXCL1, and CCL2 were responsible for increased PIN cell proliferation. These three cytokines activated ERK and JNK signaling in PIN cells through a ligand-receptor interaction. However, only blockade of ERK abolished macrophage cytokines-induced cell proliferation of PIN. Overall, our results provide a mechanistic view on how macrophages activated through chronic inflammation can expedite PIN progression during prostate cancer development. The information from our work can facilitate a comprehensive understanding of prostate cancer development, which is required for improvement of current strategies for prostate cancer therapy.

Early-onset metastatic and clinically advanced prostate cancer is a distinct clinical and molecular entity characterized by increased TMPRSS2-ERG fusions.

BACKGROUND: Men with early-onset prostate cancer are at increased risk for cancer-related mortality, yet the prevalence and spectrum of molecular alterations in this patient population is unknown. Here, we analyze comprehensive genomic profiling data to characterize the molecular drivers of early-onset prostate cancer in patients with clinically advanced and metastatic disease. METHODS: Next-generation sequencing was ordered as a part of routine clinical care for 10,189 patients with prostate cancer between 02/2013 and 03/2020 using commercially available comprehensive genomic profiling. RESULTS: Deidentified genomic data for 10,189 unique patients with prostate cancer were obtained (median age = 66 y, range = 34-90 y). 439 patients were ≤50 y (4.3%), 1928 patients were between ages of 51 and 59 y (18.9%), and 7822 patients were ≥60 y  (76.8%). Of metastatic biopsy sites, lymph node, liver, and bone were the most common in all groups, accounting for 60.2% of all specimens. Overall, 97.4% of patients harbored pathologic genomic alterations. The most commonly altered genes were TP53, TMPRSS2-ERG, PTEN, AR, MYC, MLL2, RAD21, BRCA2, APC, SPOP, PIK3CA, RB1, MLL3, CDK12, ATM, and CTNNB1. Patients ≤50 y harbored significantly more TMPRSS2-ERG fusions than patients ≥60 y, while AR copy number alterations as well as SPOP and ASXL1 mutations were significantly less frequent. CONCLUSIONS: Clinically advanced and metastatic early-onset prostate cancer is a distinct clinical subgroup with characteristic genomic alterations including increased frequency of TMPRSS2-ERG fusions and fewer AR, SPOP, and ASXL1 alterations.

FIREWORKS: a bottom-up approach to integrative coessentiality network analysis.

Genetic coessentiality analysis, a computational approach which identifies genes sharing a common effect on cell fitness across large-scale screening datasets, has emerged as a powerful tool to identify functional relationships between human genes. However, widespread implementation of coessentiality to study individual genes and pathways is limited by systematic biases in existing coessentiality approaches and accessibility barriers for investigators without computational expertise. We created FIREWORKS, a method and interactive tool for the construction and statistical analysis of coessentiality networks centered around gene(s) provided by the user. FIREWORKS incorporates a novel bias reduction approach to reduce false discoveries, enables restriction of coessentiality analyses to custom subsets of cell lines, and integrates multiomic and drug-gene interaction datasets to investigate and target contextual gene essentiality. We demonstrate the broad utility of FIREWORKS through case vignettes investigating gene function and specialization, indirect therapeutic targeting of "undruggable" proteins, and context-specific rewiring of genetic networks.

Activated ALK Cooperates with N-Myc via Wnt/ß-Catenin Signaling to Induce Neuroendocrine Prostate Cancer.

Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer with poor prognosis, and there is a critical need for novel therapeutic approaches. NEPC is associated with molecular perturbation of several pathways, including amplification of MYCN. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase involved in the pathogenesis of neuroblastoma and other malignancies where it cooperates with N-Myc. We previously identified the first case of ALK F1174C-activating mutation in a patient with de novo NEPC who responded to the ALK inhibitor, alectinib. Here, we show that coactivation of ALK and N-Myc (ALK F1174C/N-Myc) is sufficient to transform mouse prostate basal stem cells into aggressive prostate cancer with neuroendocrine differentiation in a tissue recombination model. A novel gene signature from the ALK F1174C/N-Myc tumors was associated with poor outcome in multiple human prostate cancer datasets. ALK F1174C and ALK F1174C/N-Myc tumors displayed activation of the Wnt/ß-catenin signaling pathway. Chemical and genetic ALK inhibition suppressed Wnt/ß-catenin signaling and tumor growth in vitro in NEPC and neuroblastoma cells. ALK inhibition cooperated with Wnt inhibition to suppress NEPC and neuroblastoma proliferation in vitro and tumor growth and metastasis in vivo. These findings point to a role for ALK signaling in NEPC and the potential of cotargeting the ALK and Wnt/ß-catenin pathways in ALK-driven tumors. Activated ALK and N-Myc are well known drivers in neuroblastoma development, suggesting potential similarities and opportunities to elucidate mechanisms and therapeutic targets in NEPC and vice versa. SIGNIFICANCE: These findings demonstrate that coactivation of ALK and N-Myc induces NEPC by stimulating the Wnt/ß-catenin pathway, which can be targeted therapeutically.

Posttranslational regulation of FOXA1 by Polycomb and BUB3/USP7 deubiquitin complex in prostate cancer.

Forkhead box protein A1 (FOXA1) is essential for androgen-dependent prostate cancer (PCa) growth. However, how FOXA1 levels are regulated remains elusive and its therapeutic targeting proven challenging. Here, we report FOXA1 as a nonhistone substrate of enhancer of zeste homolog 2 (EZH2), which methylates FOXA1 at lysine-295. This methylation is recognized by WD40 repeat protein BUB3, which subsequently recruits ubiquitin-specific protease 7 (USP7) to remove ubiquitination and enhance FOXA1 protein stability. They functionally converge in regulating cell cycle genes and promoting PCa growth. FOXA1 is a major therapeutic target of the inhibitors of EZH2 methyltransferase activities in PCa. FOXA1-driven PCa growth can be effectively mitigated by EZH2 enzymatic inhibitors, either alone or in combination with USP7 inhibitors. Together, our study reports EZH2-catalyzed methylation as a key mechanism to FOXA1 protein stability, which may be leveraged to enhance therapeutic targeting of PCa using enzymatic EZH2 inhibitors.

Pim1 promotes human prostate cancer cell tumorigenicity and c-MYC transcriptional activity.

BACKGROUND: The serine/threonine kinase PIM1 has been implicated as an oncogene in various human cancers including lymphomas, gastric, colorectal and prostate carcinomas. In mouse models, Pim1 is known to cooperate with c-Myc to promote tumorigenicity. However, there has been limited analysis of the tumorigenic potential of Pim1 overexpression in benign and malignant human prostate cancer cells in vivo. METHODS: We overexpressed Pim1 in three human prostate cell lines representing different disease stages including benign (RWPE1), androgen-dependent cancer (LNCaP) and androgen-independent cancer (DU145). We then analyzed in vitro and in vivo tumorigenicity as well as the effect of Pim1 overexpression on c-MYC transcriptional activity by reporter assays and gene expression profiling using an inducible MYC-ER system. To validate that Pim1 induces tumorigenicity and target gene expression by modulating c-MYC transcriptional activity, we inhibited c-MYC using a small molecule inhibitor (10058-F4) or RNA interference. RESULTS: Overexpression of Pim1 alone was not sufficient to convert the benign RWPE1 cell to malignancy although it enhanced their proliferation rates when grown as xenografts in vivo. However, Pim1 expression enhanced the in vitro and in vivo tumorigenic potentials of the human prostate cancer cell lines LNCaP and DU145. Reporter assays revealed increased c-MYC transcriptional activity in Pim1-expressing cells and mRNA expression profiling demonstrated that a large fraction of c-MYC target genes were also regulated by Pim1 expression. The c-MYC inhibitor 10058-F4 suppressed the tumorigenicity of Pim1-expressing prostate cancer cells. Interestingly, 10058-F4 treatment also led to a reduction of Pim1 protein but not mRNA. Knocking-down c-MYC using short hairpin RNA reversed the effects of Pim1 on Pim1/MYC target genes. CONCLUSION: Our results suggest an in vivo role of Pim1 in promoting prostate tumorigenesis although it displayed distinct oncogenic activities depending on the disease stage of the cell line. Pim1 promotes tumorigenicity at least in part by enhancing c-MYC transcriptional activity. We also made the novel discovery that treatment of cells with the c-MYC inhibitor 10058-F4 leads to a reduction in Pim1 protein levels.