TMPRSS2-ERG fusions confer efficacy of enzalutamide in an in vivo bone tumor growth model.

BACKGROUND: Castrate Resistant Prostate Cancer (CRPC) is an advanced disease resistant to systemic traditional medical or surgical castration, and resistance is primarily attributed to reactivation of AR through multiple mechanisms. TMPRSS2-ERG fusions have been shown to regulate AR signaling, interfere with pro-differentiation functions, and mediate oncogenic signaling. We have recently shown that ERG regulates intra-tumoral androgen synthesis and thereby facilitates AR function in prostate cancer cells. We hypothesize that enzalutamide treatment will be more effective in cells/tumors with TMPRSS2-ERG translocations because these tumors have increased AR signaling. METHODS: ERG knockdown was performed with VCaP cells using lentiviral infections to generate VCaP ERGshRNA cells and control VCaP scr cells with scrambled shRNA. Cell-growth analysis was performed to determine the effect of enzalutamide. Reverse transcription, quantitative real-time PCR (RT-qPCR) was used to determine the expression of AR responsive genes. Luciferase tagged VCaP scr and shRNA infected cells were used in an intra-tibial animal model for bone tumor growth analysis and enzalutamide treatment used to inhibit AR signaling in bone tumors. Western blotting analyzed VCaP bone tumor samples for ERG, AR, AKR1C3 and HSD3B1 and HSD3B2 expression. RESULTS: Enzalutamide inhibited the growth of VCaP scr cells more effectively than shERG cells. Analysis of AR responsive genes shows that Enzalutamide treatment at 5 micromolar concentration inhibited by 85-90% in VCaP Scr cells whereas these genes were inhibited to a lesser extent in VCaP shERG cells. Enzalutamide treatment resulted in severe growth inhibition in VCaP scr shRNA cells compared to VCaP shERG cells. In bone tumor growth experiment, VCaP ERG shRNA cells grew at slower than VCaP scr shRNA cells. Androgen biosynthetic enzyme expression is lower VCaP shERG bone tumors compared to VCaP scr shRNA bone tumors and enzalutamide inhibited the enzyme expression in both types of tumors. CONCLUSIONS: These data suggest that ERG transcription factor regulates androgen biosynthetic enzyme expression that enzalutamide treatment is more effective against VCaP bone tumors with an intact ERG expression, and that knocking down ERG in VCaP cells leads to a lesser response to enzalutamide therapy. Thus, ERG expression status in tumors could help stratify patients for enzalutamide therapy.

Pharmacological targeting of CXCL12/CXCR4 signaling in prostate cancer bone metastasis.

BACKGROUND: The CXCL12/CXCR4 axis transactivates HER2 and promotes intraosseous tumor growth. To further explore the transactivation of HER2 by CXCL12, we investigated the role of small GTP protein Gαi2 in Src and HER2 phosphorylation in lipid raft membrane microdomains and the significance of CXCR4 in prostate cancer bone tumor growth. METHODS: We used a variety of methods such as lipid raft isolation, invasion assays, an in vivo model of intratibial tumor growth, bone histomorphometry, and immunohistochemistry to determine the role of CXCR4 signaling in lipid raft membrane microdomains and effects of targeting of CXCR4 for bone tumor growth. RESULTS: We determined that (a) CXCL12/CXCR4 transactivation of EGFR and HER2 is confined to lipid raft membrane microdomains, (b) CXCL12 activation of HER2 and Src is mediated by small GTP proteins in lipid rafts, (c) inhibition of the CXCL12/CXCR4 axis through plerixafor abrogates the initial establishment of tumor growth without affecting the growth of established bone tumors, and (d) inhibition of EGFR signaling through gefitinib leads to inhibition of established bone tumor growth. CONCLUSIONS: These data suggest that lipid raft membrane microdomains are key sites for CXCL12/CXCR4 transactivation of HER2 via small GTP binding protein Gαi2 and Src kinase. The initial establishment of prostate cancer is supported by the endosteal niche, and blocking the CXCL12/CXCR4 axis of this niche along with its downstream signaling severely compromises initial establishment of tumors in the bone microenvironment, whereas expanding bone tumors are sensitive only to the members of growth factor receptor inhibition.

Targeting CXCR4 with CTCE-9908 inhibits prostate tumor metastasis.

BACKGROUND: CXCL12/CXCR4 transactivation of epidermal growth factor family receptors in lipid raft membrane microdomains on cell surface is thought to mediate tumor growth and subsequent development of metastatic disease. CTCE-9908 is a known inhibitor of CXCR4. Herein, we tested the efficacy of CTCE-9908 in inhibiting prostate cancer cell growth, invasion, and metastasis. METHODS: We used a panel of in vitro assays utilizing human prostate cancer cell lines and an in vivo orthotopic prostate cancer model to assess the anti-tumoral activity of CTCE-9908. RESULTS: We demonstrated that (a) CTCE-9908 treatment resulted in no significant change in the growth of PC-3 and C4-2B cells; (b) 50 µg/ml of CTCE-9908 inhibited the invasive properties of PC-3 cells; (c) 25 mg/kg of CTCE-9908 did not alter primary tumor growth but it did significantly reduce total tumor burden in the animal including the growth of prostate and soft tissue metastases to lymph node and distant organ tissues. Histological analysis showed that CTCE-9908 treatment resulted in tumor necrosis in primary prostate tumors and no significant change in proliferation of tumor cells as measured by Ki-67 staining; (d) CTCE-9908 inhibited the tumor angiogenesis as measured by CD34 positive vessels in tumors. CONCLUSIONS: These data suggest that CXCR4 inhibition by CTCE-9908 decreases the invasion potential in vitro, which then translated to a reduction of tumor spread with associated reduction in angiogenesis. Hence, CTCE-9908 may prove to be an efficacious novel agent to prevent and treat the spread of metastatic prostate cancer.

PTEN loss mediated Akt activation promotes prostate tumor growth and metastasis via CXCL12/CXCR4 signaling.

INTRODUCTION: The chemokine CXCL12, also known as SDF-1, and its receptor, CXCR4, are overexpressed in prostate cancers and in animal models of prostate-specific PTEN deletion, but their regulation is poorly understood. Loss of the tumor suppressor PTEN (phosphatase and tensin homolog) is frequently observed in cancer, resulting in the deregulation of cell survival, growth, and proliferation. We hypothesize that loss of PTEN and subsequent activation of Akt, frequent occurrences in prostate cancer, regulate the CXCL12/CXCR4 signaling axis in tumor growth and bone metastasis. METHODS: Murine prostate epithelial cells from PTEN+/+, PTEN+/-, and PTEN-/- (prostate specific knockdown) mice as well as human prostate cancer cell lines C4-2B, PC3, and DU145 were used in gene expression and invasion studies with Akt inhibition. Additionally, HA-tagged Akt1 was overexpressed in DU145, and tumor growth in subcutaneous and intra-tibia bone metastasis models were analyzed. RESULTS: Loss of PTEN resulted in increased expression of CXCR4 and CXCL12 and Akt inhibition reversed expression and cellular invasion. These results suggest that loss of PTEN may play a key role in the regulation of this chemokine activity in prostate cancer. Overexpression of Akt1 in DU145 resulted in increased CXCR4 expression, as well as increased proliferation and cell cycle progression. Subcutaneous injection of these cells also resulted in increased tumor growth as compared to neo controls. Akt1 overexpression reversed the osteosclerotic phenotype associated with DU145 cells to an osteolytic phenotype and enhanced intra-osseous tumor growth. CONCLUSIONS: These results suggest the basis for activation of CXCL12 signaling through CXCR4 in prostate cancer driven by the loss of PTEN and subsequent activation of Akt. Akt1-associated CXCL12/CXCR4 signaling promotes tumor growth, suggesting that Akt inhibitors may potentially be employed as anticancer agents to target expansion of PC bone metastases.

Adaptor proteins mediate CXCR4 and PI4KA crosstalk in prostate cancer cells and the significance of PI4KA in bone tumor growth.

The chemokine receptor, CXCR4 signaling regulates cell growth, invasion, and metastasis to the bone-marrow niche in prostate cancer (PCa). Previously, we established that CXCR4 interacts with phosphatidylinositol 4-kinase IIIα (PI4KIIIα encoded by PI4KA) through its adaptor proteins and PI4KA overexpressed in the PCa metastasis. To further characterize how the CXCR4-PI4KIIIα axis promotes PCa metastasis, here we identify CXCR4 binds to PI4KIIIα adaptor proteins TTC7 and this interaction induce plasma membrane PI4P production in prostate cancer cells. Inhibiting PI4KIIIα or TTC7 reduces plasma membrane PI4P production, cellular invasion, and bone tumor growth. Using metastatic biopsy sequencing, we found PI4KA expression in tumors correlated with overall survival and contributes to immunosuppressive bone tumor microenvironment through preferentially enriching non-activated and immunosuppressive macrophage populations. Altogether we have characterized the chemokine signaling axis through CXCR4-PI4KIIIα interaction contributing to the growth of prostate cancer bone metastasis.

A Novel Interaction between Chemokine and Phosphoinositide Signaling in Metastatic Prostate Cancer.

Prostate cancer commonly metastasizes to bone due to its favorable microenvironment for cell growth and survival. Currently, the standard of care for metastatic prostate cancer is medical castration in conjunction with chemotherapeutic agents and newer anti-androgen/androgen receptor therapies. While these therapies aim to improve the quality of life in patients with advanced disease, resistance to these therapies is inevitable prompting the development of newer therapies to contain disease progression. The CXCL12/CXCR4 axis has previously been shown to be involved in prostate cancer cell homing to bone tissue, and new investigations found a novel interaction of Phosphatidyl Inositol 4 kinase IIIa (PI4KA) downstream of chemokine signaling. PI4KA phosphorylates at the 4th position on phosphatidylinositol (PI), to produce PI4P and is localized to the plasma membrane (PM). At the PM, PI4KA provides precursors for the generation of PI(4,5)P2, and PI(3,4,5)P3 and helps maintain PM identity through the recruitment of lipids and signaling proteins. PI4KA is recruited to the PM through evolutionarily conserved adaptor proteins, and in PC cells, CXCR4 binds with adaptor proteins to recruit PI4KA to the PM. The objective of this review is to summarize our understanding of the role that phosphatidyl inositol lipid messengers in cancer cells.

Adaptor proteins mediate CXCR4 and PI4KA crosstalk in prostate cancer cells and the significance of PI4KA in bone tumor growth.

The chemokine receptor, CXCR4 signaling regulates cell growth, invasion, and metastasis to the bone-marrow niche in prostate cancer (PCa). Previously, we established that CXCR4 interacts with phosphatidylinositol 4-kinase IIIα (PI4KIIIα encoded by PI4KA) through its adaptor proteins and PI4KA overexpressed in the PCa metastasis. To further characterize how the CXCR4-PI4KIIIα axis promotes PCa metastasis, here we identify CXCR4 binds to PI4KIIIα adaptor proteins TTC7 and this interaction induce plasma membrane PI4P production in prostate cancer cells. Inhibiting PI4KIIIα or TTC7 reduces plasma membrane PI4P production, cellular invasion, and bone tumor growth. Using metastatic biopsy sequencing, we found PI4KA expression in tumors correlated with overall survival and contributes to immunosuppressive bone tumor microenvironment through preferentially enriching non-activated and immunosuppressive macrophage populations. Altogether we have characterized the chemokine signaling axis through CXCR4-PI4KIIIα interaction contributing to the growth of prostate cancer bone metastasis.

Dynamic expression of SNAI2 in prostate cancer predicts tumor progression and drug sensitivity.

Prostate cancer is a highly heterogeneous disease, understanding the crosstalk between complex genomic and epigenomic alterations will aid in developing targeted therapeutics. We demonstrate that, even though snail family transcriptional repressor 2 (SNAI2) is frequently amplified in prostate cancer, it is epigenetically silenced in this disease, with dynamic changes in SNAI2 levels showing distinct clinical relevance. Integrative clinical data from 18 prostate cancer cohorts and experimental evidence showed that gene fusion between transmembrane serine protease 2 (TMPRSS2) and ETS transcription factor ERG (ERG) (TMPRSS2-ERG fusion) is involved in the silencing of SNAI2. We created a silencer score to evaluate epigenetic repression of SNAI2, which can be reversed by treatment with DNA methyltransferase inhibitors and histone deacetylase inhibitors. Silencing of SNAI2 facilitated tumor cell proliferation and luminal differentiation. Furthermore, SNAI2 has a major influence on the tumor microenvironment by reactivating tumor stroma and creating an immunosuppressive microenvironment in prostate cancer. Importantly, SNAI2 expression levels in part determine sensitivity to the cancer drugs dasatinib and panobinostat. For the first time, we defined the distinct clinical relevance of SNAI2 expression at different disease stages. We elucidated how epigenetic silencing of SNAI2 controls the dynamic changes of SNAI2 expression that are essential for tumor initiation and progression and discovered that restoring SNAI2 expression by treatment with panobinostat enhances dasatinib sensitivity, indicating a new therapeutic strategy for prostate cancer.

Pro-inflammatory cytokines and chemokines initiate multiple prostate cancer biologic pathways of cellular proliferation, heterogeneity and metastasis in a racially diverse population and underlie the genetic/biologic mechanism of racial disparity: Update.

Pro-inflammatory cytokine and chemokines genes drive prostate cancer progression and metastasis: molecular mechanism update and the science that underlies racial disparity. comprehensive review article. Isaac J. Powell, S. Chinni, S.S. Reddy, Alexander Zaslavsky, Navnath Gavande Introduction: In 2013 we reported that with the use of bioinformatics and ingenuity pathway network analysis we were able to identify functional driver genes that were differentially expressed among a large population of African American men (AAM) and European American men (EAM). Pro-inflammatory cytokine genes were found to be more interactive and more expressed among AAM and have been found to be functional drivers of aggressive prostate cancer (CaP) and aggressiveness in other solid tumors. We examined these genes and biological pathways initiated by these cytokines in primary CaP tissue. Method We unravel the gene network and identified biologic pathways that impacted activation of the androgen receptor, mesenchymal epithelial transition (invasion) and chemokines associated with metastasis in the CaP tissue from 639 radical prostatectomy specimens. Results Biologic pathways identified by unraveling pro-inflammatory genes from our network, more expressed among AAM compared to EAM, were tumor necrosis factor (TNF), IL1b, IL6, and IL8. IL6 and IL8 are downstream of TNF activity and are known activators of androgen receptor and through mediators promote CaP cell proliferation. TNF and IL1b mediate tumor cell invasiveness through the activation of MMP (matrix metalloproteinase) which down regulates E-Cadherin to initiate epithelial mesenchymal transition which allows cells to become invasive in the microenvironment. Ultimately our network analysis indicates that TNF and IL1b activate CXCR4 receptor on CaP cells, which facilitates metastatic progression reportedly by binding to CXCL12 on lipid rafts and tumor implantation in the bone marrow. Conclusion Our retrospective biologic mechanistic model reveals a set of pro-inflammatory cytokines and chemokines that drive CaP aggressiveness, tumor heterogeneity, progression and metastasis. A prospective multi-institutional study needs to be conducted for clinical validation as well consideration of targeted therapy.

Clinical Efficacy of Enzalutamide vs Bicalutamide Combined With Androgen Deprivation Therapy in Men With Metastatic Hormone-Sensitive Prostate Cancer: A Randomized Clinical Trial.

Importance: Black patients have been underrepresented in prospective clinical trials of advanced prostate cancer. This study evaluated the efficacy of enzalutamide compared with bicalutamide, with planned subset analysis of Black patients with metastatic hormone-sensitive prostate cancer (mHSPC), which is a disease state responsive to androgen deprivation therapy (ADT). Objective: To compare the efficacy of enzalutamide vs bicalutamide in combination with ADT in men with mHSPC, with a subset analysis of Black patients. Design, Setting, and Participants: In this randomized clinical trial, a phase 2 screening design enabled a nondefinitive comparison of the primary outcome by treatment. Patients were stratified by race (Black or other) and bone pain (present or absent). Accrual of at least 30% Black patients was required. This multicenter trial was conducted at 4 centers in the US. Men with mHSPC with no history of seizures and adequate marrow, renal, and liver function were eligible. Data analysis was performed from February 2019 to March 2020. Interventions: Participants were randomized 1:1 to receive oral enzalutamide (160 mg daily) or bicalutamide (50 mg daily) in addition to ADT. Main Outcomes and Measures: The primary end point was the 7-month prostate-specific antigen (PSA) response (SMPR) rate, a previously accepted surrogate for overall survival (OS) outcome. Secondary end points included adverse reactions, time to PSA progression, and OS. Results: A total of 71 men (median [range] age, 65 [51-86] years) were enrolled; 29 (41%) were Black, 41 (58%) were White, and 1 (1%) was Asian. Thirty-six patients were randomized to receive enzalutamide, and 35 were randomized to receive bicalutamide. Twenty-six patients (37%) had bone pain and 37 patients (52%) had extensive disease. SMPR was achieved in 30 of 32 patients (94%; 95% CI, 80%-98%) taking enzalutamide and 17 of 26 patients (65%; 95% CI, 46%-81%) taking bicalutamide (P = .008) (difference, 29%; 95% CI, 5%-50%). Among Black patients, the SMPR was 93% (95% CI, 69%-99%) among those taking enzalutamide and 42% (95% CI, 19%-68%) among those taking bicalutamide (P = .009); among non-Black patients, the SMPR was 94% (95% CI, 74%-99%) among those taking enzalutamide and 86% (95% CI, 60%-96%) among those taking bicalutamide. The 12-month PSA response rates were 84% with enzalutamide and 34% with bicalutamide. Conclusions and Relevance: The findings of this randomized clinical trial comparing enzalutamide with bicalutamide suggest that enzalutamide is associated with improved outcomes compared with bicalutamide, in terms of the rate and duration of PSA response, in Black patients with mHSPC. Trial Registration: ClinicalTrials.gov Identifier: NCT02058706.

CXCL12/CXCR4 transactivates HER2 in lipid rafts of prostate cancer cells and promotes growth of metastatic deposits in bone.

Chemokines and their receptors function in migration and homing of cells to target tissues. Recent evidence suggests that cancer cells use a chemokine receptor axis for metastasis formation at secondary sites. Previously, we showed that binding of the chemokine CXCL12 to its receptor CXCR4 mediated signaling events resulting in matrix metalloproteinase-9 expression in prostate cancer bone metastasis. A variety of methods, including lipid raft isolation, stable overexpression of CXCR4, cellular adhesion, invasion assays, and the severe combined immunodeficient-human bone tumor growth model were used. We found that (a) CXCR4 and HER2 coexist in lipid rafts of prostate cancer cells; (b) the CXCL12/CXCR4 axis results in transactivation of the HER2 receptor in lipid rafts of prostate cancer cells; (c) Src kinase mediates CXCL12/CXCR4 transactivation of HER2 in prostate cancer cells; (d) a pan-HER inhibitor desensitizes CXCR4-induced transactivation and subsequent matrix metalloproteinase-9 secretion and invasion; (e) lipid raft-disrupting agents inhibited raft-associated CXCL12/CXCR4 transactivation of the HER2 and cellular invasion; (f) overexpression of CXCR4 in prostate cancer cells leads to increased HER2 phosphorylation and migratory properties of prostate cancer cells; and (g) CXCR4 overexpression enhances bone tumor growth and osteolysis. These data suggest that lipid rafts on the cell membrane are the key site for CXCL12/CXCR4-induced HER2 receptor transactivation. This transactivation contributes to enhanced invasive signals and metastatic growth in the bone microenvironment.

A novel cross-talk between CXCR4 and PI4KIIIα in prostate cancer cells.

Chemokine signaling regulates cell migration and tumor metastasis. CXCL12, a member of the chemokine family, and its receptor, CXCR4, a G protein coupled receptor (GPCR), are key mediators of prostate-cancer (PC) bone metastasis. In PC cells androgens activate CXCR4 gene expression and receptor signaling on lipid rafts, which induces protease expression and cancer cell invasion. To identify novel lipid-raft-associated CXCR4 regulators supporting invasion/metastasis, we performed a SILAC-based quantitative proteomic analysis of lipid-rafts derived from PC3 stable cell lines with overexpression or knockdown of CXCR4. This analysis identified the evolutionarily conserved phosphatidylinositol 4-kinase IIIα (PI4KIIIα), and SAC1 phosphatase that dephosphorylates phosphatidylinositol-4-phosphate as potential candidate CXCR4 regulators. CXCR4 interacted with PI4KIIIα membrane targeting machinery recruiting them to the plasma membrane for PI4P production. Consistent with this interaction, PI4KIIIα was found tightly linked to the CXCR4 induced PC cell invasion. Thus, ablation of PI4KIIIα in CXCR4-expressing PC3 cells reduced cellular invasion in response to a variety of chemokines. Immunofluorescence microscopy in CXCR4-expressing cells revealed localized production of PI4P on the invasive projections. Human tumor studies documented increased PI4KIIIα expression in metastatic tumors vs. the primary tumor counterparts, further supporting the PI4KIIIα role in tumor metastasis. Furthermore, we also identified an unexpected function of PI4KIIIα in GPCR signaling where CXCR4 regulates PI4KIIIα activity and mediate tumor metastasis. Altogether, our study identifies a novel cross-talk between PI4KIIIα and CXCR4 in promoting tumor metastasis and suggests that PI4KIIIα pharmacological targeting may have therapeutic benefit for advanced prostate cancer patients.

Quantitative assessment of small intraosseous prostate cancer burden in SCID mice using fluorescence imaging.

BACKGROUND: Experimental bone metastases are typically analyzed when the skeletal tumor burden is large enough to be detected by imaging or histology. By this time, the bone microenvironment is usually destroyed, preventing useful analysis of tumor-bone interactions. METHODS: Small intraosseous tumors generated by intratibial injection of C4-2B prostate cancer cells transfected with green fluorescent protein (GFP) were assessed using in vivo and ex vivo fluorescence imaging, radiography, histology, and fluorometric analysis of bone lysates. RESULTS: Ex vivo fluorescence imaging and fluorometric analysis were capable of detecting tiny bone tumors as early as 10 days after injection. Ex vivo fluorescence imaging allowed simple quantification of small skeletal tumor burden and was useful in measuring the effect of systemic therapy. CONCLUSIONS: Ex vivo fluorescence imaging is a sensitive and easy method to quantify small skeletal tumor burden. This technique allows investigation of tumor-bone interactions while the bone microanatomy is still intact.

Proteases, growth factors, chemokines, and the microenvironment in prostate cancer bone metastasis.

The arrival of cancer cells in the marrow upsets the delicate homeostatic nature of the bone microenvironment. Cell surface or secreted factors brought in by cancer cells perturb the web-like communication network between different bone cell types and bone matrix. Chemokines not only attract cancer cells from the circulation into the marrow, they also stimulate a cell signaling process leading to attachment, invasion, and further stimulation of bone matrix turnover. Cancer cell surface-associated proteases have also been associated with tumor growth and bone matrix turnover. Recent data indicate that autocrine proteolytic shedding of cell surface chemokines further promotes osteoclastogenesis. Proteases also contribute to autocrine and paracrine shedding of growth factors, another mechanism of promoting growth and expansion of the metastatic deposit. Studies of the bone microenvironment have thus revealed multiple potential targets of intervention with regard to the expanding metastatic deposit.

Tissue inhibitor of metalloproteinase-3 induces apoptosis in prostate cancer cells and confers increased sensitivity to paclitaxel.

Prostate cancer is the most common cancer and the second leading cause of cancer-related death in American men. To investigate the possible usefulness of tissue inhibitor of metalloproteinase-3 (TIMP-3) in prostate cancer gene therapy, we used an adenovirus expressing TIMP-3 to assess its role as an apoptosis trigger in highly metastatic prostate cancer cell lines PC-3 and DU-145. We showed that TIMP-3 alone induced apoptotic cell death which was triggered by mitochondrion-mediated caspase-3 activation. In combination treatment, we found that adenovirus-mediated expression of TIMP-3 greatly sensitised prostate cancer cells to chemotherapeutic drug paclitaxel, indicating a superadditive or synergistic effect of TIMP-3 and cytostatic treatment on prostate cancer cell death. The proper combination of adenovirus-mediated expression of TIMP-3 with conventional chemotherapeutic drug(s) could have potential benefits in treating highly metastatic prostate cancer.

Classification-based quantitative analysis of stable isotope labeling by amino acids in cell culture (SILAC) data.

BACKGROUND AND OBJECTIVE: Stable isotope labeling by amino acids in cell culture (SILAC) is a practical and powerful approach for quantitative proteomic analysis. A key advantage of SILAC is the ability to simultaneously detect the isotopically labeled peptides in a single instrument run and so guarantee relative quantitation for a large number of peptides without introducing any variation caused by separate experiment. However, there are a few approaches available to assessing protein ratios and none of the existing algorithms pays considerable attention to the proteins having only one peptide hit. METHODS: We introduce new quantitative approaches to dealing with SILAC protein-level summary using classification-based methodologies, such as Gaussian mixture models with EM algorithms and its Bayesian approach as well as K-means clustering. In addition, a new approach is developed using Gaussian mixture model and a stochastic, metaheuristic global optimization algorithm, particle swarm optimization (PSO), to avoid either a premature convergence or being stuck in a local optimum. RESULTS: Our simulation studies show that the newly developed PSO-based method performs the best among others in terms of F1 score and the proposed methods further demonstrate the ability of detecting potential markers through real SILAC experimental data. CONCLUSIONS: No matter how many peptide hits the protein has, the developed approach can be applicable, rescuing many proteins doomed to removal. Furthermore, no additional correction for multiple comparisons is necessary for the developed methods, enabling direct interpretation of the analysis outcomes.

Selective growth regulatory and pro-apoptotic effects of DIM is mediated by AKT and NF-kappaB pathways in prostate cancer cells.

Prostate cancer is the second leading cause of cancer related deaths in men in the United States. I3C and its in vivo dimeric product, DIM, have been found to inhibit the growth of prostate cancer cells. However, the molecular mechanism(s) by which DIM elicits its effects on prostate cancer cells has not been fully elucidated. We have previously shown that I3C induces apoptosis and inhibits the activation of NF-kappaB pathway, which could be mediated via Akt signaling pathway. In this study, we investigated whether there is any cross-talk between Akt and NF-kappaB during DIM-induced apoptosis in PC-3 prostate cancer cells. We found that DIM inhibited cell growth and induced apoptosis in PC-3 prostate cancer cells but not in non-tumorigenic CRL2221 human prostate epithelial cells. DIM also inhibited EGFR expression, PI3K kinase activity, and Akt activation, and abrogated the EGF-induced activation of PI3K in prostate cancer cells. NF-kappaB DNA-binding analysis and transfection studies with Akt cDNA constructs revealed that Akt transfection resulted in the induction of NF-kappaB activity and this was inhibited by DIM treatment. DIM treatment also showed significant induction of apoptosis in non-transfected cells compared to Akt and Akt-Myr transfected prostate cancer cells. From these results, we conclude that the inhibition of Akt and NF-kappaB activity and their cross-talk is a novel mechanism by which DIM inhibits cell growth and induces apoptotic processes in prostate cancer cells but not in non-tumorigenic prostate epithelial cells.

Akt inactivation is a key event in indole-3-carbinol-induced apoptosis in PC-3 cells.

Indole-3-carbinol (I3C) is a bioactive compound present in Brassica vegetables that shows an antitumor activity in experimental animals and inhibits the growth of human cancer cells in vitro. In recent years, studies on prostate cancer (PCa) chemoprevention have been intensified, because there is a long latency for the development of clinical PCa, which makes the PCa a better target for chemoprevention. We have shown previously that I3C induces cell growth inhibition by G(1) cell cycle arrest and induces apoptosis in a dose- and time-dependent manner in PC-3 PCa cells; however, the mechanism(s) by which I3C induces apoptosis in PC-3 cells is still not clear. A cell survival pathway involving phosphatidylinositol 3'-kinase (PI3K) and Akt is known to play an important role in inhibiting apoptosis in response to growth factor signaling, which prompted us to investigate whether this pathway plays any role in I3C-induced apoptosis in PCa cells. Here we report that I3C inhibits the phosphorylation and subsequent activation of Akt kinase. In addition, I3C abrogated epidermal growth factor (EGF)-induced activation of Akt in PC-3 cells. Western blot analyses of EGF receptor showed that I3C down-regulates the EGF receptor levels and its autophosphorylation. This was also accompanied by the inhibition of EGF-induced phosphorylation of PI3K by I3C treatment. Furthermore, the known downstream modulators of the Akt/PI3K cell survival pathway, Bcl-x(L), and BAD proteins showed decreased expression after I3C treatment. From these results, we conclude that I3C-induced apoptosis is partly mediated by the inhibition of Akt activation, resulting in the alterations in the downstream regulatory molecules of Akt activation in PC-3 cells. However, further in-depth investigation is needed to establish a cause-and-effect relationship between Akt pathway and I3C effect.

CXCR4 is a novel target of cancer chemopreventative isothiocyanates in prostate cancer cells.

Isothiocyanates (ITCs) derived from cruciferous vegetables, including phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), exhibit in vivo activity against prostate cancer in a xenograft and transgenic mouse model, and thus are appealing for chemoprevention of this disease. Watercress constituent PEITC and SFN-rich broccoli sprout extract are under clinical investigations but the molecular mechanisms underlying their cancer chemopreventive effects are not fully understood. The present study demonstrates that chemokine receptor CXCR4 is a novel target of ITCs in prostate cancer cells. Exposure of prostate cancer cells (LNCaP, 22Rv1, C4-2, and PC-3) to pharmacologically applicable concentrations of PEITC, benzyl isothiocyanate (BITC), and SFN (2.5 and 5 µmol/L) resulted in downregulation of CXCR4 expression. None of the ITCs affected secretion of CXCR4 ligand (stromal-derived factor-1). In vivo inhibition of PC-3 xenograft growth upon PEITC treatment was associated with a significant decrease in CXCR4 protein level. A similar trend was discernible in the tumors from SFN-treated TRAMP mice compared with those of control mice, but the difference was not significant. Stable overexpression of CXCR4 in PC-3 cells conferred significant protection against wound healing, cell migration, and cell viability inhibition by ITCs. Inhibition of cell migration resulting from PEITC and BITC exposure was significantly augmented by RNAi of CXCR4. This study demonstrates, for the first time, that cancer chemopreventive ITCs suppress CXCR4 expression in prostate cancer cells in vitro as well as in vivo. These results suggest that CXCR4 downregulation may be an important pharmacodynamic biomarker of cancer chemopreventative ITCs in prostate adenocarcinoma.