Regulation of Molecular Biomarkers Associated with the Progression of Prostate Cancer.

Androgen receptor signaling regulates the normal and pathological growth of the prostate. In particular, the growth and survival of prostate cancer cells is initially dependent on androgen receptor signaling. Exposure to androgen deprivation therapy leads to the development of castration-resistant prostate cancer. There is a multitude of molecular and cellular changes that occur in prostate tumor cells, including the expression of neuroendocrine features and various biomarkers, which promotes the switch of cancer cells to androgen-independent growth. These biomarkers include transcription factors (TP53, REST, BRN2, INSM1, c-Myc), signaling molecules (PTEN, Aurora kinases, retinoblastoma tumor suppressor, calcium-binding proteins), and receptors (glucocorticoid, androgen receptor-variant 7), among others. It is believed that genetic modifications, therapeutic treatments, and changes in the tumor microenvironment are contributing factors to the progression of prostate cancers with significant heterogeneity in their phenotypic characteristics. However, it is not well understood how these phenotypic characteristics and molecular modifications arise under specific treatment conditions. In this work, we summarize some of the most important molecular changes associated with the progression of prostate cancers and we describe some of the factors involved in these cellular processes.

IL-6 evoked biochemical changes in prostate cancer cells.

The pro-inflammatory cytokine IL-6 has been associated with the progression of PCa to a castration-resistant phenotype. In this work, we characterized the biochemical changes evoked by IL-6 in three different models of PCa cells, including LNCaP, C4-2, and PC3. The effect of IL-6 on PCa cells was compared with the effect obtained by co-stimulation with the cAMP-inducing agent forskolin (FSK). Stimulation of LNCaP cells with IL-6 or IL-6 + FSK evoked increased expression of the neuroendocrine marker tubulin IIIß and Cav3.2 T-type Ca2+ channel subunit. PC3 cells, representing a more advanced state of PCa, had high levels of tubulin IIIß expression without any further changes observed by treatment with IL-6 or IL-6 + FSK. Elevated expression of the glucocorticoid receptor was observed in PC3, but not in LNCaP or C4-2 cells. Glucocorticoid receptor expression was not regulated by IL-6 stimulation of LNCaP or C4-2 cells. IL-6 acting alone or together with FSK evoked a significant reduction in the expression of the transcription factor REST and retinoblastoma tumor suppressor protein Rb1. In LNCaP cells, IL-6 acting alone or together with FSK had no effect on the expression of several biological markers of advanced PCa, including Aurora kinase A, valosin-containing protein, calcium-sensing receptor, calreticulin, S100A protein, and Protein S. In PC3 cells, co-treatment with IL-6 + FSK evoked increased expression of REST and S100A proteins, as well as a reduction in Protein S levels. These findings reveal a complex pattern of biochemical changes in PCa cells under the influence of IL-6.

Major differences in glycosylation and fucosyltransferase expression in low-grade versus high-grade bladder cancer cell lines.

Bladder cancer is the ninth most frequently diagnosed cancer worldwide, and there is a need to develop new biomarkers for staging and prognosis of this disease. Here we report that cell lines derived from low-grade and high-grade bladder cancers exhibit major differences in expression of glycans in surface glycoproteins. We analyzed protein glycosylation in three low-grade bladder cancer cell lines RT4 (grade-1-2), 5637 (grade-2), and SW780 (grade-1), and three high-grade bladder cancer cell lines J82COT (grade-3), T24 (grade-3) and TCCSUP (grade-4), with primary bladder epithelial cells, A/T/N, serving as a normal bladder cell control. Using a variety of approaches including flow cytometry, immunofluorescence, glycomics and gene expression analysis, we observed that the low-grade bladder cancer cell lines RT4, 5637 and SW780 express high levels of the fucosylated Lewis-X antigen (Lex, CD15) (Galß1-4(Fucα1-3)GlcNAcß1-R), while normal bladder epithelial A/T/N cells lack Lex expression. T24 and TCCSUP cells also lack Lex, whereas J82COT cells express low levels of Lex. Glycomics analyses revealed other major differences in fucosylation and sialylation of N-glycans between these cell types. O-glycans are highly differentiated, as RT4 cells synthesize core 2-based O-glycans that are lacking in the T24 cells. These differences in glycan expression correlated with differences in RNA expression levels of their cognate glycosyltransferases, including α1-3/4-fucosyltransferase genes. These major differences in glycan structures and gene expression profiles between low- and high-grade bladder cancer cells suggest that glycans and glycosyltransferases are candidate biomarkers for grading bladder cancers.

Regulation of T-type Ca2+ channel expression by interleukin-6 in sensory-like ND7/23 cells post-herpes simplex virus (HSV-1) infection.

Herpes simplex virus-type 1 (HSV-1) infection of sensory neurons may lead to a significant reduction in the expression of voltage-activated Na+ and Ca2+ channels, which can disrupt the transmission of pain information. Viral infection also results in the secretion of various pro-inflammatory cytokines, including interleukin (IL)-6. In this work, we tested whether IL-6 regulates the expression of Na+ and Ca2+ channels post-HSV-1 infection in ND7/23 sensory-like neurons. Our results demonstrate that HSV-1 infection causes a significant decrease in the protein expression of the Cav3.2 T-type Ca2+ channel subunit, despite increasing Cav3.2 mRNA synthesis. Neither Cav3.2 mRNA nor total protein content was affected by IL-6 treatment post-HSV-1 infection. In ND7/23 cells, HSV-1 infection caused a significant reduction in the expression of Na+ and T-type Ca2+ channels within 48 h. Exposure of ND7/23 cells to IL-6 for 24 h post-infection reverses the effect of HSV-1, resulting in a significant increase in T-type Ca2+ current density. However, Na+ currents were not restored by 24-h treatment with IL-6 post-HSV-1 infection of ND7/23 cells. The ability of IL-6 to increase the functional expression of T-type Ca2+ channels on the membrane was blocked by the inhibition of protein trafficking with brefeldin-A and ERK1/2 activation. These results indicate that IL-6 release following HSV-1 infection regulates the expression of T-type Ca2+ channels, which may alter the transmission of pain information.

T-type Calcium Channels in Cancer.

Although voltage-activated Ca2+ channels are a common feature in excitable cells, their expression in cancer tissue is less understood. T-type Ca2+ channels are particularly overexpressed in various cancers. Because of their activation profile at membrane potentials close to rest and the generation of a window current, T-type Ca2+ channels may regulate a variety of Ca2+-dependent cellular processes, including cell proliferation, survival, and differentiation. The expression of T-type Ca2+ channels is of special interest as a target for therapeutic interventions.

Androgen receptor signaling regulates T-type Ca2+ channel expression and neuroendocrine differentiation in prostate cancer cells.

Therapies designed to reduce androgen production or receptor activation are effective in limiting prostate tumor growth. However, prolonged treatment with anti-androgen therapies results in the progression of prostate cancers into an androgen refractory state. Neuroendocrine differentiation (NED) has been associated with the progression of prostate cancers to an androgen resistant phenotype. In this work we investigated the effect of disrupting androgen receptor signaling in promoting NED of prostate carcinoma cells and whether it is accompanied by an increase in T-type Ca2+ channel expression. The effect of disrupting androgen signaling was assessed in LNCaP and 22Rv1 prostate cancer cells following treatment with the androgen receptor blocker, bicalutamide, or hormone-depleted media. Treatment of LNCaP cells with bicalutamide or hormone-depleted media for 4-10 d evoked considerable morphological and biochemical changes consistent with NED including the development of long neurite-like processes and the expression of the neuronal marker, tubulin IIIß. PCR analysis of bicalutamide-stimulated cells revealed no significant changes in Cav3.2 mRNA. However, stimulation of LNCaP cells with bicalutamide or hormone-depleted media for 10 d evoked a significant increase in Cav3.2 protein expression and the appearance of functional T-type Ca2+ channels. Inhibition of T-type Ca2+ channel function with various pharmacological blockers disrupted the morphological differentiation of LNCaP cells. Bicalutamide-evoked expression of functional T-type Ca2+ channels in LNCaP cells promoted chemoresistance to docetaxel. These findings indicate that disruption of androgen receptor signaling in prostate cancer cells evokes increased expression of functional T-type Ca2+ channels, which may result in significant morphological and biochemical changes.

Posttranscriptional regulation of T-type Ca(2+) channel expression by interleukin-6 in prostate cancer cells.

BACKGROUND: At early stages, the growth of prostate cancers is androgen dependent. At later stages, however, the growth of prostate cancers becomes androgen independent, which leads to an increase in mortality. The switch to an androgen-refractory state is associated with neuroendocrine differentiation (NED) of prostate cancer cells. Several factors including interleukin-6 (IL-6) and increased cAMP production promote NED of prostate cancer cells. In this work we investigated whether IL-6 evoked NED of LNCaP cells results in a significant change in T-type Ca(2+) channel expression in comparison to non-stimulated LNCaP cells. METHODS: T-type Ca(2+) channel subunit Cav3.2 expression was studied using PCR analysis, western blot and whole cell recordings. Tubulin IIIß expression and neurite-like morphology was assessed to investigate the role of T-type Ca(2+) channels in the differentiation of prostate cancer cells. RESULTS: Treatment of LNCaP cells with IL-6 for 4days evokes considerable morphological and biochemical changes consistent with NED. Transcripts of the T-type Ca(2+) channel subunit Cav3.2 but not Cav3.1 or Cav3.3 are detected in IL-6 stimulated cells. Real time PCR analysis of IL-6 stimulated cells indicates no significant change in Cav3.2 mRNA expression in comparison to non-stimulated cells. LNCaP cells stimulated with IL-6 show a threefold increase in T-type Ca(2+) channel subunit Cav3.2 protein expression, suggesting that channel expression is upregulated by a posttranscriptional mechanism. Electrophysiological recordings reveal that increased Cav3.2 protein expression following IL-6 stimulation of LNCaP cells does not result in increased expression of functional channels in the membrane. Functional expression of Cav3.2 channels in LNCaP cells is facilitated by co-stimulation with IL-6 and the cAMP-stimulating agent, forskolin (FSK). Inhibition of T-type Ca(2+) channel activity in IL-6 stimulated LNCaP cells prevents the development of morphological characteristics consistent with NED. CONCLUSIONS: These results indicate that the functional expression of T-type Ca(2+) channels is regulated by the interplay of multiple factors in LNCaP cells.

Regulation of T-type calcium channel expression by sodium butyrate in prostate cancer cells.

Several cellular mechanisms contribute to the neuroendocrine differentiation of prostate cancer cells, including exposure to sodium butyrate (NaBu), a naturally occurring salt of the short chain fatty acid n-butyric acid. NaBu belongs to a class of histone deacetylase inhibitors with potential anticancer function. T-type calcium channel expression constitutes an important route for calcium influx in tumor cells that may trigger changes in cell proliferation and differentiation. In this work we investigated the role NaBu on the differentiation of lymph node carcinoma of the prostate (LNCaP) cells and its effect on T-type Ca(2+) channel expression. NaBu stimulates the morphological and molecular differentiation of LNCaP cells. Stimulation of LNCaP cells with NaBu evokes a significant increase in the expression of the Cav3.2 T-type channel subunits. Furthermore, the increased Cav3.2 expression promotes membrane insertion of T-type Ca(2+) channels capable of generating fast inactivating Ca(2+) currents, sensitive to 100µM Ni(2+) ions. Inhibition of T-type Ca(2+) channel function reduces the outgrowth of neurite-like processes in LNCaP cells. NaBu-evoked expression of T-type Ca(2+) channels is also involved in the regulation of cell viability. Inhibition of T-type Ca(2+) channels causes a significant reduction in the viability of LNCaP cells treated with 1mM NaBu, suggesting that Ca(2+) influx via T-type channels can promote cell proliferation. However, increased expression of T-type Ca(2+) channels enhanced the cytotoxic effect of thapsigargin and paclitaxel on cell proliferation. These findings demonstrate that NaBu stimulates T-type Ca(2+) channel expression, thereby regulating both the morphological differentiation and growth of prostate cancer cells.

Downregulation of GluA2 AMPA receptor subunits reduces the dendritic arborization of developing spinal motoneurons.

AMPA receptors lacking the GluA2 subunit allow a significant influx of Ca(2+) ions. Although Ca(2+)-permeable AMPA receptors are a familiar feature at early stages of development, the functional significance of these receptors during the maturation of the nervous system remains to be established. Chicken lumbar motoneurons express Ca(2+)-permeable AMPA receptors at E6 but the Ca(2+) permeability of AMPA receptors decreases ∼3-fold by E11. Considering that activity-dependent changes in intracellular Ca(2+) regulates dendritic outgrowth, in this study we investigated whether downregulation of GluA2 expression during a critical period of development alters the dendritic arborization of spinal motoneurons in ovo. We use an avian replication-competent retroviral vector RCASBP (B) carrying the marker red fluorescent protein (RFP) and a GluA2 RNAi construct to downregulate GluA2 expression. Chicken embryos were infected at E2 with one of the following constructs: RCASBP(B)-RFP, RCASBP(B)-RFP-scrambled RNAi, or RCASBP(B)-RFP-GluA2 RNAi. Infection of chicken embryos at E2 resulted in widespread expression of RFP throughout the spinal cord with ≥60% of Islet1/2-positive motoneurons infected, resulting in a significant reduction in GluA2 protein expression. Downregulation of GluA2 expression had no effect on the dendritic arborization of E6 motoneurons. However, downregulation of GluA2 expression caused a significant reduction in the dendritic arborization of E11 motoneurons. Neither motoneuron survival nor maturation of network activity was affected by changes in GluA2 expression. These findings demonstrate that increased GluA2 expression and changes in the Ca(2+) permeability of AMPA receptors regulate the dendritic arborization of spinal cord motoneurons during a critical period of development.

Leukemia inhibitory factor regulates trafficking of T-type Ca2+ channels.

Neuropoietic cytokines such as ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) stimulate the functional expression of T-type Ca(2+) channels in developing sensory neurons. However, the molecular and cellular mechanisms involved in the cytokine-evoked membrane expression of T-type Ca(2+) channels are not fully understood. In this study we investigated the role of LIF in promoting the trafficking of T-type Ca(2+) channels in a heterologous expression system. Our results demonstrate that transfection of HEK-293 cells with the rat green fluorescent protein (GFP)-tagged T-type Ca(2+) channel α(1H)-subunit resulted in the generation of transient Ca(2+) currents. Overnight treatment of α(1H)-GFP-transfected cells with LIF caused a significant increase in the functional expression of T-type Ca(2+) channels as indicated by changes in current density. LIF also evoked a significant increase in membrane fluorescence compared with untreated cells. Disruption of the Golgi apparatus with brefeldin A inhibited the stimulatory effect of LIF, indicating that protein trafficking regulates the functional expression of T-type Ca(2+) channels. Trafficking of α(1H)-GFP was also disrupted by cotransfection of HEK-293 cells with the dominant-negative form of ADP-ribosylation factor (ARF)1 but not ARF6, suggesting that ARF1 regulates the LIF-evoked membrane trafficking of α(1H)-GFP subunits. Trafficking of T-type Ca(2+) channels required transient activation of the JAK and ERK signaling pathways since stimulation of HEK-293 cells with LIF evoked a considerable increase in the phosphorylation of the downstream JAK targets STAT3 and ERK. Pretreatment of HEK-293 cells with the JAK inhibitor P6 or the ERK inhibitor U0126 blocked ERK phosphorylation. Both P6 and U0126 also inhibited the stimulatory effect of LIF on T-type Ca(2+) channel expression. These findings demonstrate that cytokines like LIF promote the trafficking of T-type Ca(2+) channels.

Inhibition of electrical activity by retroviral infection with Kir2.1 transgenes disrupts electrical differentiation of motoneurons.

Network-driven spontaneous electrical activity in the chicken spinal cord regulates a variety of developmental processes including neuronal differentiation and formation of neuromuscular structures. In this study we have examined the effect of chronic inhibition of spinal cord activity on motoneuron survival and differentiation. Early spinal cord activity in chick embryos was blocked using an avian replication-competent retroviral vector RCASBP (B) carrying the inward rectifier potassium channel Kir2.1. Chicken embryos were infected with one of the following constructs: RCASBP(B), RCASBP(B)-Kir2.1, or RCASBP(B)-GFP. Infection of chicken embryos at E2 resulted in widespread expression of the viral protein marker p27 gag throughout the spinal cord. Electrophysiological recordings revealed the presence of functional Kir2.1 channels in RCASBP(B)-Kir2.1 but not in RCASBP(B)-infected embryos. Kir2.1 expression significantly reduced the generation of spontaneous motor movements in chicken embryos developing in ovo. Suppression of spontaneous electrical activity was not due to a reduction in the number of surviving motoneurons or the number of synapses in hindlimb muscle tissue. Disruption of the normal pattern of activity in chicken embryos resulted in a significant downregulation in the functional expression of large-conductance Ca(2+)-dependent K(+) channels. Reduction of spinal cord activity also generates a significant acceleration in the inactivation rate of A-type K(+) currents without any significant change in current density. Kir2.1 expression did not affect the expression of voltage-gated Na(+) channels or cell capacitance. These experiments demonstrate that chronic inhibition of chicken spinal cord activity causes a significant change in the electrical properties of developing motoneurons.

Extrinsic regulation of T-type Ca(2+) channel expression in chick nodose ganglion neurons.

Functional expression of T-type Ca(2+) channels is developmentally regulated in chick nodose neurons. In this study we have tested the hypothesis that extrinsic factors regulate the expression of T-type Ca(2+) channels in vitro. Voltage-gated Ca(2+) currents were measured using whole-cell patch clamp recordings in E7 nodose neurons cultured under various conditions. Culture of E7 nodose neurons for 48 h with a heart extract induced the expression of T-type Ca(2+) channels without any significant effect on HVA currents. T-type Ca(2+) channel expression was not stimulated by survival promoting factors such as BDNF. The stimulatory effect of heart extract was mediated by a heat-labile, trypsin-sensitive factor. Various hematopoietic cytokines including CNTF and LIF mimic the stimulatory effect of heart extract on T-type Ca(2+) channel expression. The stimulatory effect of heart extract and CNTF requires at least 12 h continuous exposure to reach maximal expression and is not altered by culture of nodose neurons with the protein synthesis inhibitor anisomycin, suggesting that T-type Ca(2+) channel expression is regulated by a posttranslational mechanism. Disruption of the Golgi apparatus with brefeldin-A inhibits the stimulatory effect of heart extract and CNTF suggesting that protein trafficking regulates the functional expression of T-type Ca(2+) channels. Heart extract- or CNTF-evoked stimulation of T-type Ca(2+) channel expression is blocked by the Jak/STAT and MAP kinase blockers, AG490 and U0126, respectively. This study provides new insights into the electrical differentiation of placode-derived sensory neurons and the role of extrinsic factors in regulating the functional expression of Ca(2+) channels.

Akt activation is necessary for growth factor-induced trafficking of functional K(Ca) channels in developing parasympathetic neurons.

The protein kinase Akt is a crucial regulator of neuronal survival and apoptosis. Here we show that Akt activation is necessary for mobilization of large-conductance K(Ca) channels in ciliary ganglion (CG) neurons evoked by beta-neuregulin-1 (NRG1) and transforming growth factor-beta1 (TGFbeta1). Application of NRG1 to embryonic day 9 (E9) CG neurons increased Akt phosphorylation, as observed previously for TGF(beta)1. NRG1- and TGF(beta)1-evoked stimulation of K(Ca) is blocked by inhibitors of PI3K by overexpression of a dominant-negative form of Akt, by overexpression of CTMP, an endogenous negative regulator of Akt, and by application of the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO). Conversely, overexpression of a constitutively-active form of Akt was sufficient by itself to increase mobilization of functional K(Ca) channels. NRG1 and TGF(beta)1 evoked an Akt-dependent increase in cell-surface SLO alpha-subunits. These procedures have no effect on voltage-activated Ca2+ currents. Thus Akt plays an essential role in the developmental regulation of excitability in CG neurons.

Activity- and target-dependent regulation of large-conductance Ca2+-activated K+ channels in developing chick lumbar motoneurons.

The functional expression of large-conductance (BK-type) Ca2+-activated K+ (K(Ca)) channels was examined in developing chick lumbar motoneurons (LMNs) between embryonic day 6 (E6) and E13 using patch-clamp recording techniques. The macroscopic K(Ca) current of E13 LMNs is inhibited by iberiotoxin and resistant to apamin. The average macroscopic K(Ca) density was low before E8 and increased 3.3-fold by E11, with an additional 1.8-fold increase occurring by E13. BK-type K(Ca) channels could not be detected in inside-out patches from E8 LMNs but were readily detected at E11. The density of voltage-activated Ca2+ currents did not change between E8 and E11. Surgical ablation of target tissues at E5 caused a significant reduction in average K(Ca) density in LMNs measured at E11. Conversely, chronic in ovo administration of d-tubocurarine, which causes an increase in motoneuron branching on the surface of the muscle target tissue, evoked a 1.8-fold increase in average LMN K(Ca) density measured at E11. Electrical activity also contributed to developmental regulation of LMN K(Ca) density. A significant reduction in E11 K(Ca) density was found after chronic in ovo treatment with the neuronal nicotinic antagonist mecamylamine or the GABA receptor agonist muscimol, agents that reduce activation of LMNs in ovo. Moreover, 3 d exposure to depolarizing concentrations of external K+ to LMNs cultured at E8 caused an increase in K(Ca) expression. Conversely, tetrodotoxin caused a decrease in K(Ca) expression in cultured E8 LMNs developing for 3 d in the presence of neurotrophic factors that promote neuronal survival in the absence of target tissues.