Mitochondrial proteome: altered cytochrome c oxidase subunit levels in prostate cancer

Laser capture microdissection was combined with reverse phase protein lysate arrays to quantitatively analyze the ratios of mitochondrial encoded cytochrome c oxidase subunits to nuclear encoded cytochrome c oxidase subunits, and to correlate the ratios with malignant progression in human prostate tissue specimens. Cytochrome c oxidase subunits I-III comprise the catalytic core of the enzyme and are all synthesized from mitochondrial DNA. The remaining subunits (IV-VIII) are synthesized from cellular nuclear DNA. A significant (P < 0.001, 30/30 prostate cases) shift in the relative concentrations of nuclear encoded cytochrome c oxidase subunits IV, Vb, and VIc compared to mitochondrial encoded cytochrome c oxidase subunits I and II was noted during the progression of prostate cancer from normal epithelium through premalignant lesions to invasive carcinoma. Significantly, this shift was discovered to begin even in the premalignant stage. Reverse phase protein lysate array-based observations were corroborated with immunohistochemistry, and extended to a few human carcinomas in addition to prostate. This analysis points to a role for nuclear DNA encoded mitochondrial proteins in carcinogenesis; underscoring their potential as targets for therapy while highlighting the need for full characterization of the mitochondrial proteome.     

前列腺癌雄激素受体和PI3K/Akt信号通路相互作用研究进展

前列腺癌的发生、进展依赖于雄激素,因此去势手术成为治疗晚期前列腺癌的标准疗法。但是去势后大多前列腺癌最终将转化为雄激素非依赖性前列腺癌,甚至进展为激素难治性前列腺癌,使得肿瘤的进展不受低水平雄激素的影响。即使如此,大多数激素非依赖性前列腺癌,依然阳性表达雄激素受体。因而雄激素受体在前列腺癌发生发展中起着重要作用。而PI3K/Akt信号通路能够通过维持细胞生存、抑制细胞凋亡、促进细胞代谢及血管生成等促进前列腺癌进展。本综述旨在总结前人研究,阐述雄激素受体和PI3K/Akt信号通路之间相互作用关系。研究表明Akt信号通路能够正性或者负性调控AR蛋白表达、蛋白的稳定性及其转录活性,从而维持细胞的生存、代谢。而AR即可以通过基因转录途径抑制Akt活化又能通过非转录基因途径激活Akt及其下游蛋白。因此,AR和Akt信号通路相互协同促进前列腺癌的发生及其向雄激素非依赖性前列腺癌进展。     

Addressing the effects of Salmonella internalization in host cell signaling on a reverse‐phase protein array

Through acute enteric infection, Salmonella invades host enterocytes and reproduces intracellularly into specialized vacuolae. This involves changes in host cell signaling elicited by bacterial proteins delivered via type III secretion systems (TTSS). One of the two TTSSs of Salmonella enterica serovar Typhimurium encoded by the Salmonella pathogenicity island‐1, triggers bacterial internalization. Among the effector proteins translocated by this TTSS, the GTPase modulator SopE/E2 and the phosphoinositide phosphatase SigD are known to play key roles in these processes. To better understand their contribution to re‐programming host cell pathways, we used ZeptoMARK reverse‐phase protein array technology, which allows printing 32‐sample lysate arrays that can be analyzed with phospho‐specific antibodies to evaluate the phosphorylation of signaling proteins. Lysates were obtained at different times after infection of HeLa cells with WT, TTSS‐deficient, sopE/E2 and sigD single and double deletants, as well as different sigD Salmonella mutants. Our analysis detected activation of p38, JNK and ERK mitogen‐activated protein kinases, mainly dependent on SopE/E2, as well as SigD‐dependent phosphorylation of PKB/Akt and its targets GSK‐3β and FKHR/FoxO. This is the first time that reverse‐phase protein array technology is used in the cellular microbiology field, demonstrating its value to screen for host signaling events through bacterial infection.     

Developmental Exposure to Estrogen Alters Differentiation and Epigenetic Programming in a Human Fetal Prostate Xenograft Model

Prostate cancer is the most frequent non-cutaneous malignancy in men. There is strong evidence in rodents that neonatal estrogen exposure plays a role in the development of this disease. However, there is little information regarding the effects of estrogen in human fetal prostate tissue. This study explored early life estrogen exposure, with and without a secondary estrogen and testosterone treatment in a human fetal prostate xenograft model. Histopathological lesions, proliferation, and serum hormone levels were evaluated at 7, 30, 90, and 200-day time-points after xenografting. The expression of 40 key genes involved in prostatic glandular and stromal growth, cell-cycle progression, apoptosis, hormone receptors and tumor suppressors was evaluated using a custom PCR array. Epigenome-wide analysis of DNA methylation was performed on whole tissue, and laser capture-microdissection (LCM) isolated epithelial and stromal compartments of 200-day prostate xenografts. Combined initial plus secondary estrogenic exposures had the most severe tissue changes as revealed by the presence of hyperplastic glands at day 200. Gene expression changes corresponded with the cellular events in the KEGG prostate cancer pathway, indicating that initial plus secondary exposure to estrogen altered the PI3K-Akt signaling pathway, ultimately resulting in apoptosis inhibition and an increase in cell cycle progression. DNA methylation revealed that differentially methylated CpG sites significantly predominate in the stromal compartment as a result of estrogen-treatment, thereby providing new targets for future investigation. By using human fetal prostate tissue and eliminating the need for species extrapolation, this study provides novel insights into the gene expression and epigenetic effects related to prostate carcinogenesis following early life estrogen exposure.     

Mitogen Activated Protein kinase signal transduction pathways in the prostate

The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy.     

Clusterin enhances AKT2-mediated motility of normal and cancer prostate cells through a PTEN and PHLPP1 circuit

Clusterin (CLU) is a chaperone-like protein with multiple functions. sCLU is frequently upregulated in prostate tumor cells after chemo- or radiotherapy and after surgical or pharmacological castration. Moreover, CLU has been documented to modulate the cellular homolog of murine thymoma virus akt8 oncogene (AKT) activity. Here, we investigated how CLU overexpression influences phosphatidylinositol 3′-kinase (PI3K)/AKT signaling in human normal and cancer epithelial prostate cells. Human prostate cells stably transfected with CLU were broadly profiled by reverse phase protein array (RPPA), with particular emphasis on the PI3K/AKT pathway. The effect of CLU overexpression on normal and cancer cell motility was also tested. Our results clearly indicate that CLU overexpression enhances phosphorylation of AKT restricted to isoform 2. Mechanistically, this can be explained by the finding that the phosphatase PH domain leucine-rich repeat-containing protein phosphatase 1 (PHLPP1), known to dephosphorylate AKT2 at S474, is markedly downregulated by CLU, whereas miR-190, a negative regulator of PHLPP1, is upregulated. Moreover, we found that phosphatase and tensin homolog (PTEN) was heavily phosphorylated at the inhibitory site S380, contributing to the hyperactivation of AKT signaling. By keeping AKT2 phosphorylation high, CLU dramatically enhances the migratory behavior of prostate epithelial cell lines with different migratory and invasive phenotypes, namely prostate normal epithelial 1A (PNT1A) and prostatic carcinoma 3 (PC3) cells. Altogether, our results unravel for the first time a circuit by which CLU can switch a low migration phenotype toward a high migration phenotype, through miR-190-dependent downmodulation of PHLPP1 expression and, in turn, stabilization of AKT2 phosphorylation.     

Dual Action of miR-125b As a Tumor Suppressor and OncomiR-22 Promotes Prostate Cancer Tumorigenesis

MicroRNAs (miRs) are a novel class of small RNA molecules, the dysregulation of which can contribute to cancer. A combinatorial approach was used to identify miRs that promote prostate cancer progression in a unique set of prostate cancer cell lines, which originate from the parental p69 cell line and extend to a highly tumorigenic/metastatic M12 subline. Together, these cell lines are thought to mimic prostate cancer progression in vivo. Previous network analysis and miR arrays suggested that the loss of hsa-miR-125b together with the overexpression of hsa-miR-22 could contribute to prostate tumorigenesis. The dysregulation of these two miRs was confirmed in human prostate tumor samples as compared to adjacent benign glandular epithelium collected through laser capture microdissection from radical prostatectomies. In fact, alterations in hsa-miR-125b expression appeared to be an early event in tumorigenesis. Reverse phase microarray proteomic analysis revealed ErbB2/3 and downstream members of the PI3K/AKT and MAPK/ERK pathways as well as PTEN to be protein targets differentially expressed in the M12 tumor cell compared to its parental p69 cell. Relevant luciferase+3’-UTR expression studies confirmed a direct interaction between hsa-miR-125b and ErbB2 and between hsa-miR-22 and PTEN. Restoration of hsa-miR-125b or inhibition of hsa-miR-22 expression via an antagomiR resulted in an alteration of M12 tumor cell behavior in vitro. Thus, the dual action of hsa-miR-125b as a tumor suppressor and hsa-miR-22 as an oncomiR contributed to prostate tumorigenesis by modulations in PI3K/AKT and MAPK/ERK signaling pathways, key pathways known to influence prostate cancer progression.     

Role of epithelial cell fibroblast growth factor receptor substrate 2alpha in prostate development, regeneration and tumorigenesis

The fibroblast growth factor (FGF) regulates a broad spectrum of biological activities by activation of transmembrane FGF receptor (FGFR) tyrosine kinases and their coupled intracellular signaling pathways. FGF receptor substrate 2alpha (FRS2alpha) is an FGFR interactive adaptor protein that links multiple signaling pathways to the activated FGFR kinase. We previously showed that FGFR2 in the prostate epithelium is important for branching morphogenesis and for the acquisition of the androgen responsiveness. Here we show in mice that FRS2alpha is uniformly expressed in the epithelial cells of developing prostates, whereas it is expressed only in basal cells of the mature prostate epithelium. However, expression of FRS2alpha was apparent in luminal epithelial cells of regenerating prostates and prostate tumors. To investigate FRS2alpha function in the prostate, the Frs2alpha alleles were ablated specifically in the prostatic epithelial precursor cells during prostate development. Similar to the ablation of Fgfr2, ablation of Frs2alpha disrupted MAP kinase activation, impaired prostatic ductal branching morphogenesis and compromised cell proliferation. Unlike the Fgfr2 ablation, disrupting Frs2alpha had no effect on the response of the prostate to androgens. More importantly, ablation of Frs2alpha inhibited prostatic tumorigenesis induced by oncogenic viral proteins. The results suggest that FRS2alpha-mediated signals in prostate epithelial cells promote branching morphogenesis and proliferation, and that aberrant activation of FRS2-linked pathways might promote tumorigenesis. Thus, the prostate-specific Frs2alpha(cn) mice provide a useful animal model for scrutinizing the molecular mechanisms underlying prostatic development and tumorigenesis.     

Oxidative stress signalling in the apoptosis of Jurkat T-lymphocytes

Within the first 24 h after castration of an adult male rat, the vascular system of the ventral prostate gland undergoes a degenerative process that drastically reduces blood flow to the tissue. Since the vascular degeneration precedes the loss of the prostatic epithelium (by apoptosis), we have proposed that the onset of epithelial cell apoptosis in this tissue is caused by an ischemic/hypoxic environment resulting from the loss of blood flow. In order to further evaluate the extent to which ischemia/hypoxia might be a factor in apoptosis of the prostate epithelium after castration, we analyzed for biomarkers of cellular hypoxia in rat ventral prostates during the first 3 days following castration. Ventral prostate tissues removed from hypoxyprobe-1-treated adult male rats (uncastrated controls; surgically castrated for 24, 48 or 72 h, or sham-castrated for equivalent times) were directly analyzed for evidence of hypoxia by in situ immunohistochemical evaluation of hypoxyprobe-1 adduct formation in the prostate cells. Protein extracts from these tissues were also tested for expression of the 120 kDa hypoxia-inducible factor-1-alpha (HIF-1-alpha) protein as well as for expression of mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) proteins using a Western blot assay. The tyrosine phosphorylation status of the latter signaling molecules was also evaluated by Western blotting using anti-tyrosine phosphate antibodies. Our results showed that epithelial cells of the rat ventral prostate stained positively for hypoxyprobe-1 adducts at all times after castration, whereas cells in control tissues were unstained by this procedure. In addition, the prostatic expression of HIF-1-alpha protein was increased approximately 20-fold at 48 h after castration compared to control tissues. Finally, although prostatic MAPK and JNK protein expression was unaltered during the early period after castration, phosphorylation of the JUN kinase protein was significantly elevated, indicating that this stress-activated cellular signaling pathway becomes more active subsequent to castration. These results support our proposal that early castration-induced degeneration and constriction of the vascular system of the rat ventral prostate gland leads to reduced oxygenation of prostatic epithelial cells and the activation of hypoxic cellular signaling in these cells through upregulation of HIF-1-alpha expression and stimulation of the JUN kinase signaling pathway.     

Immunocell-array for molecular dissection of multiple signaling pathways in mammalian cells

The knowledge of signaling pathways that are triggered by physiological and pathological conditions or drug treatment is essential for the comprehension of the biological events that regulate cellular responses. Recently novel platforms based on "reverse-phase protein arrays" have proven to be useful in the study of different pathways, but they still lack the possibility to detect events in the complexity of a cellular context. We developed an "immunocell-array" of cells on chip where, upon cell plating, growing, drug treatment, and fixation, by spotting specific antibodies we can detect the localization and state of hundreds of proteins involved in specific signaling pathways. By applying this technology to mammalian cells we analyzed signaling proteins involved in the response to DNA damage and identified a chromatin remodeling pathway following bleomycin treatment. We propose our technology as a new tool for the array-based multiplexed analysis of signaling pathways in drug response screening, for the proteomics of profiling patient cells, and ultimately for the high throughput screening of antibodies for immunofluorescence applications.     

DNA methylation of E-cadherin is a priming mechanism for prostate development

In prostate and other epithelial cancers, E-cadherin (CDH1) is downregulated inappropriately by DNA methylation to promote an invasive phenotype. Though cancer frequently involves a reawakening of developmental signaling pathways, whether DNA methylation of Cdh1 occurs during organogenesis has not been determined. Here we show that DNA methylation of Cdh1 mediates outgrowth of developing prostate ducts. During the three-day gestational window leading up to and including prostate ductal initiation, Cdh1 promoter methylation increases and its mRNA and protein abundance decreases in epithelium giving rise to prostatic buds. DNA methylation is required for prostate specification, ductal outgrowth, and branching morphogenesis. All three endpoints are impaired by a DNA methylation inhibitor, which also decreases Cdh1 promoter methylation and increases Cdh1 mRNA and protein abundance. A CDH1 function-blocking antibody restores prostatic identity, bud outgrowth, and potentiates epithelial differentiation in the presence of the DNA methylation inhibitor. This is the first study to mechanistically link acquired changes in DNA methylation to the normal process of prostate organogenesis. We propose a novel mechanism whereby Cdh1 promoter methylation restricts Cdh1 abundance in developing prostate epithelium to create a permissive environment for prostatic bud outgrowth. Thus, DNA methylation primes the prostate primordium to respond to developmental cues mediating outgrowth, differentiation and maturation of the ductal network.     

Prostate tumor-stroma interaction: molecular mechanisms and opportunities for therapeutic targeting

Maintenance of cell and tissue homeostasis is dependent upon the dynamic balance of cell proliferation, differentiation, and apoptosis through interactions between cells and their microenvironment. The unique prostatic cellular phenotypes are induced and maintained by interaction between epithelium and adjacent stroma through intimate intercellular signaling pathways. In this article, we summarize current advances in the tumor-stroma interaction and its biologic and therapeutic implications. We specifically emphasize current studies of the possible factors driving the "vicious cycle" between stroma and emerging prostate tumor epithelial cells that may be responsible for carcinogenesis and metastasis to bone. Stroma responds both genotypically and phenotypically to tumor epithelium upon co-culture under 3-D conditions. Likewise, the emerging carcinoma responds to stromal signals that drive progression to malignancy. A vicious cycle mediated by soluble and insoluble molecules secreted by tumor cells and stroma appear be the critical factors supporting and sustaining tumor colonization in bone. Co-targeting tumor and stroma with therapeutic agents has yielded promising results both in pre-clinical models of prostate cancer and bony metastasis and in clinical trials of patients treated with a dual tumor and stroma targeting strategies. In conclusion, understanding and targeting the interaction of the tumor and its stromal microenvironmant may improve the prognosis, reduce the suffering and increase the survival of patients with advanced cancer metastasis.     

Antibody arrays in cancer research

Antibody arrays have valuable applications in cancer research. Many different antibody array technologies have been developed, each with particular advantages, disadvantages, and optimal applications. The methods have been demonstrated on various sample types, such as serum, plasma, and other bodily fluids; cell culture supernatants; tissue culture lysates; and resected tumor specimens. The applications to cancer research have included profiling proteins to identify candidate biomarkers, characterizing signaling pathways, and the measurement of changes in modification or expression level of cancer-related proteins. Further innovations in the methods and experimental strategies are broadening the scope of the applications and the type of information that can be gathered. These alternate formats and uses of antibody arrays include arrays to measure whole cells, arrays to measure enzyme activities, reverse phase arrays, and bead-based arrays. This article reviews the various types of antibody array methods and their applications to cancer research.     

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.     

Conditional expression of the androgen receptor induces oncogenic transformation of the mouse prostate

The androgen signaling pathway, mediated through the androgen receptor (AR), is critical in prostate tumorigenesis. However, the precise role of AR in prostate cancer development and progression still remains largely unknown. Specifically, it is unclear whether overexpression of AR is sufficient to induce prostate tumor formation in vivo. Here, we inserted the human AR transgene with a LoxP-stop-loxP (LSL) cassette into the mouse ROSA26 locus, permitting "conditionally" activated AR transgene expression through Cre recombinase-mediated removal of the LSL cassette. By crossing this AR floxed strain with Osr1-Cre (odd skipped related) mice, in which the Osr1 promoter activates at embryonic day 11.5 in urogenital sinus epithelium, we generated a conditional transgenic line, R26hAR(loxP):Osr1-Cre+. Expression of transgenic AR was detected in both prostatic luminal and basal epithelial cells and is resistant to castration. Approximately one-half of the transgenic mice displayed mouse prostatic intraepithelial neoplasia (mPIN) lesions. Intriguingly, four mice (10%) developed prostatic adenocarcinomas, with two demonstrating invasive diseases. Positive immunostaining of transgenic AR protein was observed in the majority of atypical and tumor cells in the mPIN and prostatic adenocarcinomas, providing a link between transgenic AR expression and oncogenic transformation. An increase in Ki67-positive cells appeared in all mPIN and prostatic adenocarcinoma lesions of the mice. Thus, we demonstrated for the first time that conditional activation of transgenic AR expression by Osr1 promoter induces prostate tumor formation in mice. This new AR transgenic mouse line mimics the human disease and can be used for study of prostate tumorigenesis and drug development.     

Utility of reverse phase protein arrays: applications to signalling pathways and human body arrays.

Protein microarrays offer a new means by which to conduct quantitative profiling of disease-associated proteins. The knowledge gained may provide novel strategies for early detection, diagnosis and therapeutic intervention. A variety of sophisticated approaches, including gene arrays, sequencing consortiums and large-scale two-dimensional gel electrophoresis, continue to generate lists of proteins potentially linked to disease aetiology and progression. The challenge is to evaluate quantitatively promising lead protein candidates using matched normal and diseased cell populations. In contrast to the antibody array, the reverse phase protein microarrays (RPPA) do not require labelling of cellular protein lysates, and constitute a sensitive high throughput platform for marker screening, pathophysiology investigation and therapeutic monitoring. In this paper, examples will be provided using RPPAs in the study of the apoptotic signalling cascade and in the evaluation of the expression of organ-specific protein makers using microdissected human organ cell lysates configured as 'human body arrays'.