Transformation Of Prostate Epithelial Cells Research Articles

CEACAM expression in an in-vitro prostatitis model.

Prostatitis is an inflammatory disease of the prostate gland, which affects 2-16% of men worldwide and thought to be a cause for prostate cancer (PCa) development. Carcinoembryogenic antigen-related cell adhesion molecules (CEACAMs) are deregulated in inflammation and in PCa. The role of CEACAMs in prostate inflammation and their possible contribution to the malignant transformation of prostate epithelial cells is still elusive. In this study, we investigated the expression of CEACAMs in an in-vitro prostatitis model and their potential role in malignant transformation of prostate epithelial cells. Normal prostate epithelial RWPE-1 cells were treated with pro-inflammatory cytokines to achieve an inflammatory state of the cells. The expression of CEACAMs and their related isoforms were analyzed. Additionally, the expression levels of selected CEACAMs were correlated with the expression of malignancy markers and the migratory properties of the cells. This study demonstrates that the pro-inflammatory cytokines, tumor necrosis factor alpha (TNFα) and interferon-gamma (IFNγ), induce synergistically an up-regulation of CEACAM1 expression in RWPE-1 cells, specifically favoring the CEACAM1-L isoform. Furthermore, overexpressed CEACAM1-L is associated with the deregulated expression of JAK/STAT, NFκB, and epithelial-mesenchymal transition (EMT) genes, as well as an increased cell migration. We postulate that CEACAM1 isoform CEACAM1-4L may synergistically contribute to inflammation-induced oncogenesis in the prostate.

Enhanced Succinate Oxidation with Mitochondrial Complex II Reactive Oxygen Species Generation in Human Prostate Cancer.

The transformation of prostatic epithelial cells to prostate cancer (PCa) has been characterized as a transition from citrate secretion to citrate oxidation, from which one would anticipate enhanced mitochondrial complex I (CI) respiratory flux. Molecular mechanisms for this transformation are attributed to declining mitochondrial zinc concentrations. The unique metabolic properties of PCa cells have become a hot research area. Several publications have provided indirect evidence based on investigations using pre-clinical models, established cell lines, and fixed or frozen tissue bank samples. However, confirmatory respiratory analysis on fresh human tissue has been hampered by multiple difficulties. Thus, few mitochondrial respiratory assessments of freshly procured human PCa tissue have been published on this question. Our objective is to document relative mitochondrial CI and complex II (CII) convergent electron flow to the Q-junction and to identify electron transport system (ETS) alterations in fresh PCa tissue. The results document a CII succinate: quinone oxidoreductase (SQR) dominant succinate oxidative flux model in the fresh non-malignant prostate tissue, which is enhanced in malignant tissue. CI NADH: ubiquinone oxidoreductase activity is impaired rather than predominant in high-grade malignant fresh prostate tissue. Given these novel findings, succinate and CII are promising targets for treating and preventing PCa.

Abstract 2999: Activation of NFKB is responsible for defective autophagy in cadmium-induced transformation of prostate cells

Abstract Background: Evidence from the epidemiological, laboratory and clinical studies suggest that chronic exposure to cadmium (Cd) leads to the development and progression of prostate cancer (CaP). Previously, we reported defective autophagy due to upregulation of Placenta Specific 8 (Plac8) (a phagosome and lysosome fusion regulator) is responsible for Cd-induced transformation of normal prostate epithelial cells. This study mechanistically dissects the upstream signaling that regulates Plac8 function, which drives defective autophagy during Cd-induced transformation. Methods: To examine the molecular interaction between NFκB and Plac8, we developed NFκB/Plac8 overexpression and knockdown in normal prostate epithelial cells (RWPE-1) and cadmium transformed prostate epithelial cells lines (CTPE). In addition, we performed phenotypic, and molecular analysis including promoter-based studies, western blot, and in vivo analysis to determine the interaction between Plac8 and p65 in Cd-exposed prostate epithelial cells. Results: Our results confirmed that overexpression of Plac8 in normal prostate epithelial cells mimics Cd-transformed prostate cells (CTPE) and induces defective autophagy by regulating autophagy-related genes (ATGs) and an enhanced autophagic flux. Conversely, either NFκB or Plac8 knockdown in CTPE cells abrogates autophagy signaling. Further, Cd-exposure failed to induce defective autophagy signaling in transformed cells. Silencing p65 either by genetically or pharmacological inhibitors downregulated Plac8 expression and its survival function in CTPE cells. Hence, we investigated the interaction between NFκB and Plac8 by promoter-based analysis. We found NFκB binding site in the Plac8 promoter, and mutating NFκB binding sites in the Plac8 promoter showed a decline in luciferase activity confirming our hypothesis that NFκB activation is necessary for Plac8 activation and autophagy signaling in CTPE cells. Finally, we confirmed that silencing Plac8 significantly inhibited the tumor growth of CTPE cells compared to vehicle control. Our ongoing studies on xenotransplanted stably knockdown of p65 in CTPE cells may confirm that NFκB activation is responsible for Plac8 function and tumor growth in mice models. Conclusion: In conclusion, our results suggest that NFκB transcriptionally regulates Plac8 function, which drives pro-survival autophagy signaling in Cd-transformed prostate epithelial cells. Citation Format: Vaibhav Shukla, Ashish Tyagi, Venkatesh Kolluru, Uttara Saran, Balaji Chandrasekaran, Murali K. Ankem, Chendil Damodaran. Activation of NFKB is responsible for defective autophagy in cadmium-induced transformation of prostate cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2999.

Dietary Pterostilbene for MTA1-Targeted Interception in High-Risk Premalignant Prostate Cancer.

Prostate cancer remains one of the most prevalent cancers in aging men. Active surveillance subpopulation of patients with prostate cancer includes men with varying cancer risk categories of precancerous disease due to prostatic intraepithelial neoplasia (PIN) heterogeneity. Identifying molecular alterations associated with PIN can provide preventable measures through finding novel pharmacologic targets for cancer interception. Targeted nutritional interception may prove to be the most appropriate chemoprevention for intermediate- and high-risk active surveillance patients. Here, we have generated two prostate-specific transgenic mouse models, one overexpressing MTA1 (R26MTA1 ) and the other overexpressing MTA1 on the background of Pten heterozygosity (R26MTA1 ; Pten+/f ), in which we examined the potential chemopreventive efficacy of dietary pterostilbene. We show that MTA1 promotes neoplastic transformation of prostate epithelial cells by activating cell proliferation and survival, leading to PIN development. Moreover, MTA1 cooperates with PTEN deficiency to accelerate PIN development by increasing cell proliferation and MTA1-associated signaling. Further, we show that mice fed with a pterostilbene-supplemented diet exhibited more favorable histopathology with decreased severity and number of PIN foci accompanied by reduced proliferation, angiogenesis, and inflammation concomitant to reduction in MTA1 and MTA1-associated CyclinD1, Notch2, and oncogenic miR-34a and miR-22 levels. PREVENTION RELEVANCE: Developing novel interceptive strategies for prostate cancer chemoprevention is a paramount goal in clinical oncology. We offer preclinical evidence for the potential of pterostilbene as a promising natural agent for MTA1-targeted interceptive strategy in future cancer prevention trials towards protecting select patients with prostate cancer under active surveillance from developing cancer.

Circulating extracellular vesicles release oncogenic miR-424 in experimental models and patients with aggressive prostate cancer

Extracellular vesicles (EVs) are relevant means for transferring signals across cells and facilitate propagation of oncogenic stimuli promoting disease evolution and metastatic spread in cancer patients. Here, we investigated the release of miR-424 in circulating small EVs or exosomes from prostate cancer patients and assessed the functional implications in multiple experimental models. We found higher frequency of circulating miR-424 positive EVs in patients with metastatic prostate cancer compared to patients with primary tumors and BPH. Release of miR-424 in small EVs was enhanced in cell lines (LNCaPabl), transgenic mice (Pb-Cre4;Ptenflox/flox;Rosa26ERG/ERG) and patient-derived xenograft (PDX) models of aggressive disease. EVs containing miR-424 promoted stem-like traits and tumor-initiating properties in normal prostate epithelial cells while enhanced tumorigenesis in transformed prostate epithelial cells. Intravenous administration of miR-424 positive EVs to mice, mimicking blood circulation, promoted miR-424 transfer and tumor growth in xenograft models. Circulating miR-424 positive EVs from patients with aggressive primary and metastatic tumors induced stem-like features when supplemented to prostate epithelial cells. This study establishes that EVs-mediated transfer of miR-424 across heterogeneous cell populations is an important mechanism of tumor self-sustenance, disease recurrence and progression. These findings might indicate novel approaches for the management and therapy of prostate cancer.

Stromal-epithelial interactions in prostate cancer: Overexpression of PAGE4 in stromal cells inhibits the invasive ability of epithelial cells.

It is now widely recognized that carcinoma-associated fibroblasts which are believed to be myofibroblasts, promote the transformation of prostate epithelial cells to cancer cells, enhance their proliferation and invasiveness, and induce the acquisition of resistance to cancer therapy and immune evasiveness. Prostate-associated gene 4 (PAGE4) is an intrinsically disordered protein that is remarkably prostate-specific. PAGE4 is also a stress-response protein that functions as a transcriptional regulator and is upregulated in early-stage prostate cancer (PCa) and its precursor lesions. However, PAGE4 is downregulated in high-grade PCa and metastatic disease. Here, we show that PAGE4 is highly expressed in the stromal cells surrounding the cancer-adjacent "normal" glands and low-grade PCa lesions but not in lesions proximal to high-grade PCa. Overexpression of PAGE4 in a stromal cell line inhibits the migration and invasion of PCa epithelial cells in multiple coculture systems. PAGE4 overexpression also inhibits the downregulation of E-cadherin in PCa epithelial cells when cocultured with stromal cells. Furthermore, signaling via tumor necrosis factor-α and transforming growth factor-β pathways is decreased in the stromal cells overexpressing PAGE4 suggesting that PAGE4 appears to play a protective role against disease progression by perturbing interactions between epithelial cells and stromal cells in PCa. Taken together, these findings support previous observations that upregulation of PAGE4 in PCa correlates with a better prognosis and highlight PAGE4 as a novel therapeutic target for early-stage "low-risk" disease.

Centrosome loss results in an unstable genome and malignant prostate tumors.

Localized, nonindolent prostate cancer (PCa) is characterized by large-scale genomic rearrangements, aneuploidy, chromothripsis, and other forms of chromosomal instability (CIN), yet how this occurs remains unclear. A well-established mechanism of CIN is the overproduction of centrosomes, which promotes tumorigenesis in various mouse models. Therefore, we developed a single-cell assay for quantifying centrosomes in human prostate tissue. Surprisingly, centrosome loss-which has not been described in human cancer-was associated with PCa progression. By chemically or genetically inducing centrosome loss in nontumorigenic prostate epithelial cells, mitotic errors ensued, producing aneuploid, and multinucleated cells. Strikingly, transient or chronic centrosome loss transformed prostate epithelial cells, which produced highly proliferative and poorly differentiated malignant tumors in mice. Our findings suggest that centrosome loss could create a cellular crisis with oncogenic potential in prostate epithelial cells.

Abstract 4277: Inhibition of autophagy (Plac8) prevents malignant transformation of cadmium induced prostate carcinogenesis

Abstract Introduction: Cadmium (Cd) exposure is associated with an increased risk of prostate cancer (CaP). Previously we reported that Cd induces Plac8 expression may be responsible for defective autophagy which facilitates the transformation of prostate epithelial cells. The goal of this study is to decipher the molecular signaling responsible for defective autophagy by plac8 and whether inhibition of plac8 facilitates pro-apoptotic machinery in chronically exposed -Cd in prostate epithelial cells. Methods: Overexpression or silencing Plac8 in chronically exposed Cd in RWPE-1 cells or cadmium, transformed prostate epithelial (CTPE) cells were subjected to cell viability, apoptosis, autophagy functional studies, Western blot and in vivo analyses. For statistical analysis, data were analyzed using Student’s‘t’ test with a p-value less than 0.05 considered significant. Results: Our results suggest that during cellular transformation, Cd exposure induced Plac8 expression, which caused defective-autophagy that resulted in the proliferation of damaged cells. Inhibition of Plac8 resulted in suppression of pro-survival machinery and concomitantly induced severe endoplasmic reticulum (ER) stress that resulted in caspase-mediated cell death in Cd-exposed cells. More specifically, inhibition of Plac8 by shRNA resulted in induction of ER stress markers (PREK/ATF4) which facilitate caspase-mediated cell death in Cd- transformed prostate epithelial cells. Activation of NFKB is one of the downstream events of Plac8 that transcriptionally regulates NFKB activation in Cd-transformed cells. We identified, Plac8 binding sites in NFKB promoter. Hence inhibition of plac8 resulted in the complete shutdown of NFKB mediated pro-survival signaling in Cd-transformed prostate epithelial cells. While analyzing autophagy signaling network, we found induction of Atg family proteins, however, in the absence of Plac8, the induction of autophagy fails to protect the Cd-transformed cells. Similar results were observed in plac8 silenced cd-transformed cells in xenotransplanted mice, where Cd-transformed cells induced aggressive tumors, and Plac8 knockdown significantly inhibited the tumor growth. While ATF4, PLac8, NFKB were not expressed in normal human prostate tissues, their expression was significantly increased in BPH followed by CaP specimens; normal prostate < BPH << Gleason 6-7 CaP < Gleason 8-10 CaP. Cd levels were much higher in tumor tissues (21.43 μg/g) of CaP patients than in healthy controls (1.12 μg/g). The levels also correlated with Plac8 expression. These results demonstrate the clinical significance of Cd levels and of Plac8 expression as plausible drivers of Cd-induced transformation in prostate carcinogenesis. Conclusions: These results suggest that Plac8 may be an essential component in the Cd-induced transformation of normal prostate epithelial cells to a cancerous state. Citation Format: Venkatesh Kolluru, Ashish Tyagi, Balaji Chandrasekaran, Murali K. Ankem, Chendil Damodaran. Inhibition of autophagy (Plac8) prevents malignant transformation of cadmium induced prostate carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4277.

Прогностическое значение простатспецифического антигена при определении показаний к первичной биопсии предстательной железы

To investigate the diagnostic accuracy of PSA and its derivatives in detecting benign and malignant diseases of the prostate after an initial prostate biopsy. The current study analyzed 65 initial biopsies performed due to an elevated serum PSA level and compared them with the results of extended PSA testing with the use of PSA derivatives. The histological findings consistent with PCa were found in about 30% of initial biopsies performed due to elevated serum levels of total PSA. The incidence of histologically confirmed PCa increased with age, and 70-79 year old men were more likely to have it than 60-69 years old patients. PSA density in 85% of PCa cases exceeded that for benign prostatic hyperplasia (BPH). In all PCa patients, PHI and free /total PSA fraction were greater than 25 and less than 0.15, respectively. In most patients with BPH, the PHI and free /total PSA fraction did not exceed 25 and were greater than 0.15. 1. Initial biopsy triggered by elevated serum level of total PSA allows detection of PCa in about 30% of cases. 2. The age of 60 years and older should be regarded as a risk factor for PCa. The probability of malignant transformation of prostate epithelial cells in men over 70 years of age is greater than in 60-69-year-olds. 3. The density of PSA in men with morphologically verified PCa is statistically significantly greater than that in men with BPH and the threshold value in 85% of cases. 4. Determination of PHI and free/total PSA fraction can improve the accuracy of predicting malignant lesions of the prostate and adjust the indications for histological examination, reduce the number of unnecessary biopsies. 5. When defining indications for initial prostate biopsy in cases with serum PSA varying from 2 to 10 ng/ml, PSA derivatives should be used, including PSA density, prostate health index, and the free /total PSA fraction.

The androgen receptor malignancy shift in prostate cancer.

Androgens and the androgen receptor (AR) are necessary for the development, function, and homeostatic growth regulation of the prostate gland. However, once prostate cells are transformed, the AR is necessary for the proliferation and survival of the malignant cells. This change in AR function appears to occur in nearly every prostate cancer. We have termed this the AR malignancy shift. In this review, we summarize the current knowledge of the AR malignancy shift, including the DNA-binding patterns that define the shift, the transcriptome changes associated with the shift, the putative drivers of the shift, and its clinical implications. In benign prostate epithelial cells, the AR primarily binds consensus AR binding sites. In carcinoma cells, the AR cistrome is dramatically altered, as the AR associates with FOXA1 and HOXB13 motifs, among others. This shift leads to the transcription of genes associated with a malignant phenotype. In model systems, some mutations commonly found in localized prostate cancer can alter the AR cistrome, consistent with the AR malignancy shift. Current evidence suggests that the AR malignancy shift is necessary but not sufficient for transformation of prostate epithelial cells. Reinterpretation of prostate cancer genomic classification systems in light of the AR malignancy shift may improve our ability to predict clinical outcomes and treat patients appropriately. Identifying and targeting the molecular factors that contribute to the AR malignancy shift is not trivial but by doing so, we may be able to develop new strategies for the treatment or prevention of prostate cancer.

Induction of Plac8 promotes pro-survival function of autophagy in cadmium-induced prostate carcinogenesis

Chronic exposure to cadmium is known to be a risk factor for human prostate cancer. Despite over-whelming evidence of cadmium causing carcinogenicity in humans, the specific underlying molecular mechanisms that govern metal-induced cellular transformation remain unclear. Acute exposure (up to 72 h) to cadmium induces apoptosis in normal prostate epithelial cells (RWPE-1), while chronic exposure (>1 year) transforms these cells to a malignant phenotype (cadmium-transformed prostate epithelial cells; CTPE). Increased expression of autophagy-regulated genes; Plac8, LC3B and Lamp-1; in CTPE cells was associated with cadmium-induced transformation. Increased expression of Plac8, a regulator of autophagosome/autolysosome fusion, facilitates the pro-survival function of autophagy and upregulation of pAKT(ser473) and NF-κβ, to allow CTPE to proliferate. Likewise, inhibition of Plac8 suppresses CTPE cell growth. Additionally, overexpression of Plac8 in RWPE-1 cells induces resistance to cadmium toxicity. Pharmacological inhibitors and an inducer of autophagy failed to affect Plac8 expression and CTPE cell viability, suggesting a unique role for Plac8 in cadmium-induced prostate epithelial cell transformation. These results support a role for Plac8 as an essential component in the cadmium-induced transformation of normal prostate epithelial cells to a cancerous state.

Sustained Early Disruption of Mitochondrial Function Contributes to Arsenic-Induced Prostate Tumorigenesis.

Arsenic is a well-known human carcinogen that affects millions of people worldwide, but the underlying mechanisms of carcinogenesis are unclear. Several epidemiological studies have suggested increased prostate cancer incidence and mortality due to exposure to arsenic. Due to lack of an animal model of arsenic-induced carcinogenesis, we used a prostate epithelial cell culture model to identify a role for mitochondria in arsenic-induced prostate cancer. Mitochondrial morphology and membrane potential was impacted within a few hours of arsenic exposure of non-neoplastic prostate epithelial cells. Chronic arsenic treatment induced mutations in mitochondrial genes and altered mitochondrial functions. Human non-neoplastic prostate epithelial cells continuously cultured for seven months in the presence of 5 µM arsenite showed tumorigenic properties in vitro and induced tumors in SCID mice, which indicated transformation of these cells. Protein and mRNA expression of subunits of mtOXPHOS complex I were decreased in arsenic-transformed cells. Alterations in complex I, a main site for reactive oxygen species (ROS) production as well as increased expression of ROS-producing NOX4 in arsenic-transformed cells suggested a role of oxidative stress in tumorigenic transformation of prostate epithelial cells. Whole genome cGH array analyses of arsenic-transformed prostate cells identified extensive genomic instability. Our study revealed mitochondrial dysfunction induced oxidative stress and decreased expression of p53 in arsenic-transformed cells as an underlying mechanism of the mitochondrial and nuclear genomic instability. These studies suggest that early changes in mitochondrial functions are sustained during prolong arsenic exposure. Overall, our study provides evidence that arsenic disruption of mitochondrial function is an early and key step in tumorigenic transformation of prostate epithelial cells.

Abstract 3343: Targeting the Lin28A/B axis reverts stem cell-like phenotype and tumor-initiating properties in prostate cancer

Abstract Cancer stem cells (CSCs) represent the most tumorigenic, metastatic and therapy resistant cell subpopulation within human tumors. Current therapies target bulk tumor cells while spare the most aggressive CSC subpopulation. Understanding the mechanisms responsible for the acquisition and maintenance of the CSC phenotype will help to identify new strategies to target CSCs. In this study, we describe a link between deregulated expression of the ETS transcription factor ESE3/EHF and upregulation of Lin28A and Lin28B in prostate CSC-enriched subpopulations. Furthermore, using various cell line models and in vitro/in vivo experimental systems we demonstrate the efficacy of targeting the Lin28A/B axis for selective elimination of cancer cells with tumor-initiating and stem-like properties. Mechanistically, we found that ESE3/EHF represses transcription of Lin28A and Lin28B in normal prostate epithelial cells. Downregulation of ESE3/EHF in prostate tumors led to upregulation of Lin28A and Lin28B and consequent reduction of microRNAs (miRNAs) of the let-7 family, which exert tumor suppressor functions. These events promoted cell transformation and expansion of the prostate CSC subpopulation. Conversely, targeting Lin28A/Lin28B with small interfering RNAs (siRNAs) in transformed prostate epithelial cells and prostate cancer cell lines restored the levels of let-7 miRNAs, decreased expression of several CSC marker genes, and restrained tumor-sphere formation and self-renewal properties in vitro and tumor-initiating capability in vivo. Notably, systemic treatment with a siRNA targeting Lin28B reduced growth of prostate tumor xenografts in mice. This was associated with a significant contraction of the CSC subpopulation in tumor xenografts, as demonstrated by the reduced content of ex vivo tumor-sphere forming cells, reduced expression of CSC marker genes, and upregulation of let-7 miRNAs. Furthermore, tumor cells derived from siLin28B-treated xenografts exhibited reduced in vivo tumor-initiating and self-renewal capability, in line with a persistent loss of CSC properties. Collectively, these data establish the Lin28/let-7 axis as a critical element in malignant transformation and acquisition of tumor-initiating and stem-like properties and identify a valid therapeutic strategy to antagonize CSCs in human prostate cancer. Citation Format: Domenico Albino, Gianluca Civenni, Cecilia Dallavalle, Martina Roos, Hartmut Jahns, Jonathan Hall, Giuseppina M. Carbone, Carlo V. Catapano. Targeting the Lin28A/B axis reverts stem cell-like phenotype and tumor-initiating properties in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3343.

Abstract 5251: Chemoprevention of cadmium induced prostate carcinogenesis

Abstract Cadmium is known carcinogen that causes damages to multiple organs. Cadmium acts as an agonist and activates androgen receptor (AR) in prostate epithelial cells which in turn promotes the carcinogenesis of prostate. Studies have indicated that occupational exposure of cadmium is a risk factor for prostate cancer and hence there is an immediate need to prevent cadmium induced prostate cancer in occupational workers. Various reports have shown that inhibition of AR signaling by AR inhibitors suppressed cadmium induced prostate carcinogenesis. So, the present study focuses to identify dietary agent which can effectively prevent cadmium induced prostate cancer and to dissect the mechanism of action on malignant transformed cells. We have disclosed one such compound, 6-(3, 9-dihydroxy-2-prenylcoumestan (Psoralidin) (Pso) a non-toxic, orally bioavailable compound, which effectively targets only prostate cancer cells and innocuous to healthy cells. Our results have shown cadmium transformed prostate epithelial cells (CTPE) exhibits high proliferative and high colony forming ability in soft agar assays. However, treatment of CTPE cells with Pso resulted in growth inhibition, without causing toxicity to normal prostate epithelial cells (RWPE-1). Finally, in vivo experiments showed that oral administration of Pso (20 mg/kg/BW) effectively suppresses the growth in CTPE xenografts, at physiologically achievable doses without any toxicity. The pathological findings suggest that CTPE tumors exhibits epithelial and mesenchymal transition (EMT) phenotype and infiltrating margins into the surrounding muscle and adipose tissue. However, Psoralidin treated tumors showed a decrease in tumor size with a significant central necrosis (up to 60% of the tumor size). More specifically, margins of Psoralidin treated tumors are less infiltrative and becoming more well circumscribed. We have also noticed that the mesenchymal component is not present and the only viable tumor is the epithelial one after Pso treatment. Altogether, our results confirm that consumption of natural compounds such as Psoralidin may play an important role in the chemoprevention for cadmium induced prostate carcinogenesis. Citation Format: Suman Suman, Trinath P. Das, Houda Alatassi, Murali K. Ankem, Chendil Damodaran. Chemoprevention of cadmium induced prostate carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5251.

Opposite regulation of MDM2 and MDMX expression in acquisition of mesenchymal phenotype in benign and cancer cells.

Plasticity of cancer cells, manifested by transitions between epithelial and mesenchymal phenotypes, represents a challenging issue in the treatment of neoplasias. Both epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are implicated in the processes of metastasis formation and acquisition of stem cell-like properties. Mouse double minute (MDM) 2 and MDMX are important players in cancer progression, as they act as regulators of p53, but their function in EMT and metastasis may be contradictory. Here, we show that the EMT phenotype in multiple cellular models and in clinical prostate and breast cancer samples is associated with a decrease in MDM2 and increase in MDMX expression. Modulation of EMT-accompanying changes in MDM2 expression in benign and transformed prostate epithelial cells influences their migration capacity and sensitivity to docetaxel. Analysis of putative mechanisms of MDM2 expression control demonstrates that in the context of defective p53 function, MDM2 expression is regulated by EMT-inducing transcription factors Slug and Twist. These results provide an alternative context-specific role of MDM2 in EMT, cell migration, metastasis, and therapy resistance.

Endogenous Dach1 in cancer.

The Dachshund gene is a key component of the retinal determination gene network in Drosophila eye development. Recent studies have demonstrated an important role for the human Dachshund homologue 1 (DACH1) in tumorigenesis [1]. Mechanistic studies demonstrated that DACH1 regulates its target genes via either directly binding to specific DNA sequences within chromatin or indirectly through forming protein complex with other transcriptional factors, including c-Jun and Smad4. DACH1 restrained proliferation, migration in vitro and repressed tumor growth and metastasis in vivo. There prior observations were based on cultured cells engineering DACH1 expression. The physiological role of the endogenous DACH1 had not been determined in part because genetic deletion in the mice was periembryonic lethal. A recent study report in the May 15 issue of Cancer Research determined the role of endogenous Dach1 in the prostate using tissue specific deletion mice [2]. Using prostate cancer cell lines of distinct genetic background, DACH1 was shown to inhibit proliferation in vitro and tumor growth in vivo of both AR negative cancer cell (PC-3) and castration resistant AR positive cell (C4-2 and 22RV-1). Consistent with the prior finding that DACH1 inhibited cell cycle progression though inhibiting Cyclin D1 expression, conditional Dach1 knock out in the prostate by using Dach1 fl/fl/Probasin-Cre bi-transgenic increased the expression of cyclin D1, E and A, accompanied by enhanced DNA synthesis and reduced apoptosis [2]. Whole genomic expression profiling with functional pathway analysis identified the cytokine-cytokine receptor interaction as a key target of endogenous Dach1. CXCL family member CXCL-1, 2, 5, 6 and IL-6, 8 expressions were inhibited by DACH1 in PC-3 cell. DACH1 mRNA expression was reduced in metastatic human prostate cancer [3] and DACH1 abundance was inversely correlated with IL-6 and IL-8 (Fig. ​(Fig.1).1). In agreement, there was 1000 fold activation of IL-6 and IL-8 secretion when endogenous Dach1 was deleted in vivo from prostate epithelial cells (PEC). Functional assays, using immune neutralizing antibodies or purified recombinant cytokines to conditioned medium from wt or Dach1 KO PEC, proved that IL-6 and KC (homolog of IL-8 in mice) were the key downstream targets of Dach1 in governing cell migration. In addition, various single oncogene (c-Myc, NeuT, H-Ras or v-Src) transformed prostate epithelial cells (PEC) reduced Dach1 expression both in cultured cells and in extirpated tumor tissues [2]. Combined with previous finding that DACH1 represses ligand induced transcriptional activation of the androgen receptor and prostate cancer cellular proliferation in tissue culture [3], it is likely that DACH1 conveys distinct functions at different stages of prostate cancer onset and progression. Figure 1 Reduced expression of DACH1 activates heterotypic signaling in prostate cancer The transition from hormone-dependent to castrate-resistant prostate cancer (CRPC) is the major cause of therapeutic failure. Clinical studies have shown that increased IL-6 and IL-8 signaling correlates with CRPC and predicts poor prognosis [4]. IL-6 and IL-8 produced by prostate cancer cell promote cancer cell proliferation and invasion. Moreover, IL-8 signaling from tumor cells initiates cancer cell-stromal interaction to induce treatment resistance and angiogenesis [5]. The finding that DACH1 is a key endogenous gene that restrains cytokine signaling may have therapeutic relevance. The finding that DNA demethylation agents could restore DACH1 expression in PC-3 cells [2] suggests targeting DACH1 may be practical. DACH1 also directly suppressed IL-8 in breast cancer cells and inhibited KC-mediated lung metastasis [6]. A subsequent study in lung cancer has also demonstrated that DACH1 can suppress the secretion of CXCL5, thereby reducing CXCL5-mediated proliferation, migration and invasion. Moreover, as with prostate cancer, a reverse relationship between DACH1 and CXCL5 was reported in tumor samples and low DACH1 correlated with reduced survival in lung cancer patients [7]. Thus, DACH1 governs cell fate through intracellular transcriptional regulation and also through heterotypic signaling that determines cancer-stromal interaction, a key hallmark of cancer [8].

Tumor-stroma metabolic relationship based on lactate shuttle can sustain prostate cancer progression

BackgroundCancer cell adopts peculiar metabolic strategies aimed to sustain the continuous proliferation in an environment characterized by relevant fluctuations in oxygen and nutrient levels. Monocarboxylate transporters MCT1 and MCT4 can drive such adaptation permitting the transport across plasma membrane of different monocarboxylic acids involved in energy metabolism.MethodsRole of MCTs in tumor-stroma metabolic relationship was investigated in vitro and in vivo using transformed prostate epithelial cells, carcinoma cell lines and normal fibroblasts. Moreover prostate tissues from carcinoma and benign hypertrophy cases were analyzed for individuating clinical-pathological implications of MCT1 and MCT4 expression.ResultsTransformed prostate epithelial (TPE) and prostate cancer (PCa) cells express both MCT1 and MCT4 and demonstrated variable dependence on aerobic glycolysis for maintaining their proliferative rate. In glucose-restriction the presence of L-lactate determined, after 24 h of treatment, in PCa cells the up-regulation of MCT1 and of cytochrome c oxidase subunit I (COX1), and reduced the activation of AMP-activated protein kinase respect to untreated cells. The blockade of MCT1 function, performed by si RNA silencing, determined an appreciable antiproliferative effect when L-lactate was utilized as energetic fuel. Accordingly L-lactate released by high glycolytic human diploid fibroblasts WI-38 sustained survival and growth of TPE and PCa cells in low glucose culture medium. In parallel, the treatment with conditioned medium from PCa cells was sufficient to induce glycolytic metabolism in WI-38 cells, with upregulation of HIF-1a and MCT4. Co-injection of PCa cells with high glycolytic WI-38 fibroblasts determined an impressive increase in tumor growth rate in a xenograft model that was abrogated by MCT1 silencing in PCa cells. The possible interplay based on L-lactate shuttle between tumor and stroma was confirmed also in human PCa tissue where we observed a positive correlation between stromal MCT4 and tumor MCT1 expression.ConclusionsOur data demonstrated that PCa progression may benefit of MCT1 expression in tumor cells and of MCT4 in tumor-associated stromal cells. Therefore, MCTs may result promising therapeutic targets in different phases of neoplastic transformation according to a strategy aimed to contrast the energy metabolic adaptation of PCa cells to stressful environments.

Evidence for a Novel Mechanism of the PAK1 Interaction with the Rho-GTPases Cdc42 and Rac

P21-activated kinase 1 (PAK1) is activated by binding to GTP-bound Rho GTPases Cdc42 and Rac via its CRIB domain. Here, we provide evidence that S79 in the CRIB domain of PAK1 is not directly involved in this binding but is crucial for PAK1 activation. S79A mutation reduces the binding affinity of PAK1 for the GTPases and inhibits autophosphorylation and kinase activity of PAK1. Thus, this mutation abrogates the ability of PAK1 to induce changes in cell morphology and motility and to promote malignant transformation of prostate epithelial cells. We also show that growth of the prostate cancer cell line PC3 is inhibited by the treatment of a PAK1-inhibiting peptide comprising 19 amino acids centered on S79, but not by the PAK1 peptide containing the S79A mutation, and that this growth inhibition is correlated with reduced autophosphorylation activity of PAK1. Together, these findings demonstrate a significant role of S79 in PAK1 activation and provide evidence for a novel mechanism of the CRIB-mediated interaction of PAK1 with Cdc42 and Rac.

Protein kinase CK2 phosphorylates and activates p21-activated kinase 1

Activation of the p21-activated kinase 1 (PAK1) is achieved through a conformational change that converts an inactive PAK1 dimer to an active monomer. In this paper, we show that this change is necessary but not sufficient to activate PAK1 and that it is, rather, required for CK2-dependent PAK1(S223) phosphorylation that converts a monomeric PAK1 into a catalytically active form. This phosphorylation appears to be essential for autophosphorylation at specific residues and overall activity of PAK1. A phosphomimetic mutation (S223E) bypasses the requirement for GTPases in PAK1 activation, whereas the constitutive activity of the PAK1 mutant (PAK1(H83,86L)), postulated to mimic GTPase-induced structural changes, is abolished by inhibition of S223 phosphorylation. Thus, S223 is likely accessible to CK2 upon conformational changes of PAK1 induced by GTPase-dependent and GTPase-independent stimuli, suggesting that S223 phosphorylation may play a key role in the final step of the PAK1 activation process. The physiological significance of this phosphorylation is reinforced by the observations that CK2 is responsible for epidermal growth factor-induced PAK1 activation and that inhibition of S223 phosphorylation abrogates PAK1-mediated malignant transformation of prostate epithelial cells. Taken together, these findings identify CK2 as an upstream activating kinase of PAK1, providing a novel mechanism for PAK1 activation.

Immunohistochemical expression of ERG in the molecular epidemiology of fatal prostate cancer study

Gene fusions between the ERG transcription factor and the androgen-regulated gene TMPRSS2 occur in a subset of prostate cancers and contribute to transformation of prostatic epithelial cells. Prior reports have used fluorescence in situ hybridization (FISH) or quantitative PCR (QPCR) to determine the presence of TMPRSS2-ERG fusions or ERG expression, respectively. Recently, several groups have reported on immunohistochemistry (IHC) to measure ERG expression, which is much more readily performed in clinical practice. However, the prior studies examining ERG expression by IHC had small sample sizes or they failed to clarify the association of ERG protein expression with important clinico-pathological features or prostate cancer-specific mortality. To address these deficits, we evaluated ERG expression by IHC in 208 radical prostatectomy samples from the Kaiser Permanente Molecular Epidemiology of Fatal Prostate Cancer (MEFPC) study, a case-control study of prostate cancer-specific mortality. Nuclear ERG expression was seen in neoplastic prostate epithelia in 49 of the samples (23.7%). ERG expression in tumor cells was associated with higher tumor stage (OR = 2.0, 95% confidence interval 1.0-4.0, P value = 0.04). ERG immunoreactivity was positively associated with prostate cancer-specific mortality, although the confidence interval was wide (OR = 1.9, 95% confidence interval 0.88-4.0, P value = 0.10). Our results demonstrate that ERG protein expression is readily quantifiable with an existing commercial antibody. Evaluating ERG protein expression may improve our ability to identify the subset of more aggressive, invasive prostate cancers.