Significance of Increased Tissue Transglutaminase in Hormone Refractory Prostate Cancer

The progression of hormone responsive to hormone refractory prostate cancer poses a major clinical challenge in the successful treatment of prostate cancer. The hormone refractory prostate cancer cells exhibit resistance not only to castrate levels of testosterone, but also to other therapeutic modalities and hence become lethal. Currently, there is no effective treatment available for managing this cancer. These observations underscore the urgency to investigate mechanism(s) that contribute to the progression of hormone-responsive to hormone-refractory prostate cancer and to target them for improved clinical outcomes. Tissue transglutaminase (TG2) is a multifunctional pro-inflammatory protein involved in diverse physiological processes such as inflammation, tissue repair, and wound healing. Its expression is also implicated in pathological conditions such as cancer and fibrosis. Interestingly, we found that the androgen-independent prostate cancer cell lines, which lacked androgen receptor (AR) expression, contained high basal levels of tissue transglutaminase. Inversely, the cell lines that expressed androgen receptor lacked transglutaminase expression. This attracted our attention to investigate the possible role this protein may play in the progression of prostate cancer, especially in view of recent observations that its expression is linked with increased invasion, metastasis, and drug resistance in multiple cancer cell types. The results we obtained were rather surprising and revealed that stable expression of tissue transglutaminase in androgen-sensitive LNCaP prostate cancer cells rendered these cells independent of androgen for growth and survival by silencing the AR expression. The AR silencing in TG2 expressing cells (TG2-infected LNCaP and PC-3 cells) was due to TG2-induced activation of the inflammatory nuclear transcription factor-kB (NF-kB). Thus, TG2 induced NF-kB was found to directly bind to the AR promoter. Importantly, TG2 protein was specifically recruited to the AR promoter in complex with the p65 subunit of NF-kB. Moreover, TG2 expressing LNCaP and PC-3 cells exhibited epithelial-to-mesenchymal transition, as evidenced by gain of mesenchymal (such as fibronectin, vimentin, etc.) and loss of epithelial markers (such as E-cadherin, b-catenin). Taken together, these results suggested a new function for TG2 and revealed a novel mechanism that is responsible for the progression of prostate cancer to the aggressive hormone-refractory phenotype.

Characterization of osteopontin charge forms produced by osteoblasts

Osteopontin (OPN) is a highly-phosphorylated extracellular matrix protein localized in bone, kidney, placenta, T-lymphocytes, macrophages, smooth muscle of the vascular system, milk, urine, and plasma. In ROS 17/2.8 osteoblast-like osteosarcoma cells, 1,25-dihydroxyvitamin D3 [1,25(OH)2D 3] regulates OPN at the transcriptional level resulting in increased steady state mRNA levels and increased production of OPN protein, maximal at 48 hours. Using ROS 17/2.8 cells as an osteoblast model, OPN was purified from culture medium after three hour treatments of either vehicle (ethanol) or 1,25(OH)2D3 via barium citrate precipitation followed by immunoaffinity chromatography. ^ Here, further evidence of regulation of OPN by 1,25(OH)2D 3 at the posttranslational level is presented. Prior to the up-regulation of OPN at the transcriptional level, 1,25(OH)2D3 induces a shift in OPN isoelectric point (pI) detected on two-dimensional gels from pI 4.6 to pI 5.1. Loading equal amounts of [32P]-labeled OPN recovered from ROS 17/2.8 cells exposed to 1,25(OH)2D3 or vehicle alone for three hours reveals that the shift from pI 4.6 to 5.1 is the result of reduced phosphorylation. Using structural analogs to 1,25(OH) 2D3, analog AT [25-(OH)-16-ene-23-yne-D3], which triggers Ca2+ influx through voltage sensitive Ca2+ channels but does not bind to the vitamin D receptor, mimicked the OPN pI shift while analog BT [1,25(OH)2-22-ene-24-cyclopropyl-D 3], which binds to the vitamin D receptor but does not allow Ca 2+ influx, did not. Inclusion of the Ca2+ channel blocker nifedipine also blocks the charge shift conversion of OPN. Further analysis of the signaling pathway initiated by 1,25(OH)2D3 reveals that inhibition of the cyclic 3′,5′ -adenosine monophosphate-dependent kinase, protein kinase A, or inhibition of the cyclic 3′,5′-guanine monophosphate-dependent kinase, protein kinase G, also prevents the charge shift conversion. ^ Isolation of OPN from rat femurs and tibiae provides evidence for the existence of these two OPN charge forms in vivo, evidenced by differential migration on isoelectric focusing gels and sodium dodecyl sulfate-polyacrylamide gels. Peptide sequencing of rat long bone fractions revealed the presence of a presumed dentin specific protein, dentin matrix protein-1 (DMP-1). Western blot analysis confirmed the existence of DMP-1 in these fractions. ^ Using the OPN charge forms in functional assays, it was determined that the charge forms have differential roles in both cell surface and mineralization functions. In cell attachment assays and Ca2+ influx assays using PC-3 prostate cancer cells, the pI 5.1 charge form of OPN was found to permit binding and increase intracellular Ca2+ concentrations of PC-3 cells. The increase in intracellular Ca2+ concentration was found to be integrin αvβ3-dependent. In mineralization assays, the pI 4.6 charge form of OPN promoted hydroxyapatite formation, while the pI 5.1 charge form had improved Ca2+ binding ability. ^ In conclusion, these findings suggest that 1,25(OH) 2D3 regulates OPN not only at the transcriptional level, but also plays a role in determination of the OPN phosphorylation state. The latter involves a short term (less than three hours) treatment and is associated with membrane-initiated Ca2+ influx. Functional assays utilizing the two OPN charge forms reveal the dependence of OPN post-translational state on its function. ^

A study in the molecular and cellular functions of GSK3beta in prostate adenocarcinoma

Kinases are part of a complex network of signaling pathways that enable a cell to respond to changes in environmental conditions in a regulated and coordinated way. For example, Glycogen Synthase Kinase 3 beta (GSK3β) modulates conformational changes, protein-protein interaction, protein degradation, and activation of unique domains in proteins that transduce signals from the extracellular milieu to the nucleus. ^ In this project, I investigated the expression and function that GSK3β exhibits in prostate cells. The capacity of GSK3β to regulate two transcription factors (JUN and CREB), which are known to be inversely utilized in prostate tumor cells, was measured. JUN/AP1 is constitutively activated in PC-3 cells; whereas, CREB/CRE activity is ∼20 fold less than the former. GSK3β overexpression obliterates JUN/AP1 activity. With respect to CREB GSK3β increases CREB/CRE activity. Cellular levels of active GSK3β can determine whether JUN or CREB is preferentially active in the PC-3s. Theoretically, in response to a particular cellular context or stimulus, a cell may coordinate JUN and CREB function by regulating GSK3β.^ A comparison of various prostate cell lines showed that active GSK3β is less expressed in normal prostate epithelial cells than in tumor cells. Differentially expressed active (GSK3β) may correlate with progression of prostate carcinoma. If a known marker associated with carcinoma of the prostate could be shown to be regulated by GSK3β then, further study of GSK3β may lead to a better understanding of both possible prevention of the disease and improved therapy for advanced stages. ^ The androgen receptor (AR) is an intriguing phosphoprotein whose regulation is potentially determined by a variety of kinases. One of these is (GSK3β) I found that (GSK3β) is a regulator of the androgen receptor in both the unliganded and liganded states. It can inhibit AR function as measured by reporter assays. Also, GSK3β associates with the AR at the DNA binding domain because deletion constructs expressing either the n-terminus or the c-terminus (both having the DBD in common) immunoprecipitated with GSK3β. Increased understanding of how GSK3β functions in prostate cancer would provide clues into how (1) certain signal pathways are coordinated and (2) the androgen receptor may be regulated. ^

Cleavage and activation of calpain and its substrate caspase-7 in prostate cancer cells: Evidence for a role in apoptotic sensitization

Changes in the levels of intracellular calcium mediate multiple biological effects, including apoptosis, in some tumor cells. Early studies demonstrated that prostate cancer cells are highly sensitive to alterations in the levels of their intracellular calcium pools. Furthermore, it has been established that apoptosis in prostate cancer could be initiated through calcium-selective ionophores, or inhibitors of intracellular calcium pumps. High sensitivity to changes in intracellular calcium levels may therefore be exploited as a novel mechanism for controlling prostate cancer apoptotic thresholds; however, the mechanisms associated with this process are poorly understood. To investigate the role of calcium as a mediator of prostate cancer cell death and its effects on caspase activation, LNCaP and PC-3 cell response to the calcium ionophore A23187, were examined. LNCaP cells were highly sensitive to changes in intracellular calcium, and subtoxic concentrations of A23187 facilitated apoptosis initiated by cytokines (TNF or TRAIL). In contrast, PC-3 cell death was not affected by A23187 or cytokines. A23187 caused rapid and concentration-dependent activation of calpain in LNCaP (but not PC-3 cells) which correlated with cleavage of calpain substrates caspase-7 and PTP1B. Cleavage of PTP1B from a 50 kDa to 42 kDa protein correlated with its translocation from the endoplasmic reticulum to the cytosol and with inhibition of tyrosine phosphorylation. Caspase-7 was cleaved from a 35 kDa to 30 kDa protein in response to A23187 in LNCaP (but not PC-3) cells and correlated with activation of both upstream and downstream caspases. Extracts from A23187-treated LNCaP cells, or PC-3 cells transiently transfected with calpain, mediated similar processing of in vitro transcribed and translated (TNT) caspase-7. In vitro processing of caspase-7 correlated with its proteolytic activation, which was inhibited by calpain inhibitor (calpeptin) and to some degree, by caspase inhibitors (zVAD, DEVD). Together, these results suggest that calpain is directly involved in calcium-mediated apoptosis of prostate cancer cells through activation and cleavage of caspase-7 and other substrates. Loss of calpain activation may therefore play a critical role in apoptotic resistance of some prostate cancer cells. ^

Preclinical evaluation of proteasome inhibition for the treatment of prostate cancer

Prostatic carcinoma is the most prevalent cancer detected in men. Bortezomib is the first proteasome inhibitor to undergo clinical trials for several forms of cancer. Although we know this class of agent preferentially kills cancer cells, our knowledge of proteasome inhibition mechanisms of induced death is far from complete. We investigated the effects of bortezomib on the LNCaP-Pro5 (Pro5) and PC-3-Pro4 (Pro4) human prostatic adenocarcinoma cells lines. We showed a reduction in proliferation and an increase in DNA fragmentation, caspase 3 activity, and cell surface phosphatidyl serine exposure. The bortezomib-treated tumors from both cell lines were dramatically reduced, and apoptosis was induced. There was also a reduction in proliferation in the treated tumors from both cells lines. We looked at changes in the levels of the proangiogenic factors VEGF, IL-8 and bFGF in vitro and in vivo. Although there was a reduction in the levels of VEGF produced by the Pro5 cell line and tumor due to bortezomib, no similar observations were made for the other angiogenic factors or in the Pro4 cells. We investigated the effects of bortezomib on p53 in the Pro5 cell line. Bortezomib induced strong stabilization of p53. It did not promote phosphorylation on serines 15 and 24 and p53 remained bound to its inhibitor, mdm2. Nonetheless, confocal microscopy revealed that bortezomib stimulated p53 translocation to the nucleus and enhanced p53 DNA binding, accumulation of p53-dependant transcripts, and activation of a p53-responsive reporter gene. Furthermore, stable transfectants of LNCaP-Pro5 expressing the p53 inhibitor, HPV-E6, displayed reduced bortezomib-induced p53 activation and cell death. Our data shows bortezomib to induce antitumor effects in the human Pro4 and Pro5 prostatic adenocarcinoma cell lines by the direct induction of apoptosis. The drug also causes a reduction in cell proliferation and mean vessel density while modulating the secretion of proangiogenic factors. Although we show that proteasome inhibition stimulates p53 activation via a novel mechanism in Pro5 cells, it is also toxic to p53 null cells as is seen in the Pro4 line. ^

The characterization of PKC-isozyme specific EGFR-dependent Erk1/2 activation in androgen-independent prostate cancer cell lines

One growth factor receptor commonly altered during prostate tumor progression is the epidermal growth factor receptor (EGFR). EGFR signaling regulates Erk1/2 phosphorylation through multiple mechanisms. We hypothesized that PKC isozymes play a role in EGFR-dependent signaling, and that through PKC isozyme selective inhibition, EGFR-dependent Erk1/2 activation can be attenuated in AICaP cells. ^ To test the hypothesis, PKC activation was induced by 12-O-tetradecanoyi-phorbol-13-acetate (TPA) in PC-3 cells. As a result, Erk1/2 was activated similarly to what was observed upon EGF stimulation. EGF-induced Erk1/2 activation in PC-3 cells was PKC-dependent, as demonstrated through use of a selective PKC inhibitor, GF109203X. This provides evidence for PKC regulatory control over Erk1/2 signaling downstream of EGFR. Next, we demonstrated that when PKC was inhibited by GF109203X, EGF-stimulated Erk1/2 activation was inhibited in PC-3, but not DU145 cells. TPA-stimulated Erk1/2 activation was EGFR-dependent in both DU145 and PC-3 cells, demonstrated through abrogation of Erk1/2 activation by a selective EGFR inhibitor AG1478. These data support PKC control at or upstream of EGFR in AICaP cells. We observed that interfering with ligand/EGFR binding abrogated Erk1/2 signaling in TPA-stimulated cells, revealing a role for PKC upstream of EGFR. ^ Next, we determined which PKC isozymes might be responsible for Erk1/2 regulation. We first determined that human AICaP cell lines express the same PKC isozymes as those observed in clinical prostate cancer specimens (α, ϵ, &zgr;, ι and PKD). Isozyme-selective methods were employed to characterize discrete PKC isozyme function in EGFR-dependent Erk1/2 activation. Pharmacologic inhibitors implicated PKCα in TPA-induced EGFR-dependent Erk1/2 activation in both PC-3 and DU145 cells. Further, the cPKC-specific inhibitor, Gö6976 decreased viablilty of DU145 cells, providing evidence that PKCα is necessary for growth and survival. Finally, resveratrol, a phytochemical with strong cancer therapeutic potential inhibited Erk1/2 activation, and this correlated with selective inhibition of PKCα. These results demonstrate that PKC regulates pathways critical to progression of CaP cells, including those mediated by EGFR. Thus, PKC isozyme-selective targeting is an attractive therapeutic strategy, and understanding the role of specific PKC isozymes in CaP cell growth and survival may aid in development of effective, non-toxic PKC-targeted therapies. ^

Calcium mobilization from the endoplasmic reticulum to the mitochondria during apoptosis in prostate cancer

Previous studies have implicated Ca2+ fluxes in the control of apoptosis but their exact roles in regulating the process remain obscure. Because Ca2+ can serve as a signal for cytochrome c release from isolated mitochondria, we hypothesized that alterations in intracellular Ca2+ compartmentalization might serve as a release signal in whole cells undergoing apoptosis. Exposure of human PC-3 prostate adenocarcinoma cells to staurosporine or DNA damaging agent (doxorubicin) but not to anti-Fas antibody led to early release of Ca2+ from the endoplasmic reticulum and subsequent accumulation of Ca2+ within mitochondria. Both events were blocked in cells stably transfected with Bcl-2 but were not affected by treatment with the pancaspase inhibitor, zVADfmk. The effects of staurosporine were associated with re-localization of Bax from the cytosol to both endoplasmic reticular and mitochondrial membranes. Neither ER Ca 2+ pool depletion nor mitochondrial Ca2+ uptake were observed in DU-145 cells that possess a frameshift mutation in the Bax gene unless wild-type Bax was restored via adenoviral gene transfer. Cytochrome c release and downstream features of apoptosis were attenuated by treatment with an inhibitor of mitochondria) Ca2+ uptake (RU-360). Although, direct pharmacological ER Ca2+ pool emptying in cells treated with thapsigargin did not lead to early cytochrome c release, pretreatment of cells with staurosporine dramatically sensitized mitochondria to thapsigargin-induced cytochrome c release. Together, our data demonstrate that ER-to-mitochondrial Ca2+ fluxes promote cytochrome c release and apoptosis in cells exposed to some (but not all) pro-apoptosic stimuli. ^