Prostate Cancer Cell Lines C4-2B Research Articles

Activation of neural lineage networks and ARHGEF2 in enzalutamide-resistant and neuroendocrine prostate cancer and association with patient outcomes

BackgroundTreatment-emergent neuroendocrine prostate cancer (NEPC) after androgen receptor (AR) targeted therapies is an aggressive variant of prostate cancer with an unfavorable prognosis. The underlying mechanisms for early neuroendocrine differentiation are poorly defined and diagnostic and prognostic biomarkers are needed.MethodsWe performed transcriptomic analysis on the enzalutamide-resistant prostate cancer cell line C4-2B MDVR and NEPC patient databases to identify neural lineage signature (NLS) genes. Correlation of NLS genes with clinicopathologic features was determined. Cell viability was determined in C4-2B MDVR and H660 cells after knocking down ARHGEF2 using siRNA. Organoid viability of patient-derived xenografts was measured after knocking down ARHGEF2.ResultsWe identify a 95-gene NLS representing the molecular landscape of neural precursor cell proliferation, embryonic stem cell pluripotency, and neural stem cell differentiation, which may indicate an early or intermediate stage of neuroendocrine differentiation. These NLS genes positively correlate with conventional neuroendocrine markers such as chromogranin and synaptophysin, and negatively correlate with AR and AR target genes in advanced prostate cancer. Differentially expressed NLS genes stratify small-cell NEPC from prostate adenocarcinoma, which are closely associated with clinicopathologic features such as Gleason Score and metastasis status. Higher ARGHEF2, LHX2, and EPHB2 levels among the 95 NLS genes correlate with a shortened survival time in NEPC patients. Furthermore, downregulation of ARHGEF2 gene expression suppresses cell viability and markers of neuroendocrine differentiation in enzalutamide-resistant and neuroendocrine cells.ConclusionsThe 95 neural lineage gene signatures capture an early molecular shift toward neuroendocrine differentiation, which could stratify advanced prostate cancer patients to optimize clinical treatment and serve as a source of potential therapeutic targets in advanced prostate cancer.

Design, synthesis, and anti-tumor activities of novel Brevinin-1BYa peptidomimetics

Brevinin-1BYa is an amphibian skin-derived peptide that exhibits promising anti-microbial activity against gram-positive and -negative bacteria. However, the anti-tumor activity of Brevinin-1BYa remains unclear, and, more importantly, its therapeutic application is limited owing to its poor protease and reduction stability. In this study, a series of novel Brevinin-1BYa derivatives, including O-linked N-acetyl-glucosamine glyclopeptides and disulfide bond mimetics, were designed and synthesized. Additionally, their anti-tumor activity against human prostate cancer cell line C4-2B, human NSCLC cell line A549 (adenocarcinoma), and human hepatoma cells line HuH-7 was investigated. Among these, the thioether bridge substituted peptidomimetic Brevinin-1BYa-3 displayed improved reduction stability, more stable secondary structure, greater protease stability, and increased anti-tumor activity compared with the original peptide, rendering it a promising leading compound for drug development, particularly for applications against malignant tumors.

Osteoblasts Generate Testosterone From DHEA and Activate Androgen Signaling in Prostate Cancer Cells.

Bone metastasis is a complication of prostate cancer in up to 90% of men afflicted with advanced disease. Therapies that reduce androgen exposure remain at the forefront of treatment. However, most prostate cancers transition to a state whereby reducing testicular androgen action becomes ineffective. A common mechanism of this transition is intratumoral production of testosterone (T) using the adrenal androgen precursor dehydroepiandrosterone (DHEA) through enzymatic conversion by 3β- and 17β-hydroxysteroid dehydrogenases (3βHSD and 17βHSD). Given the ability of prostate cancer to form blastic metastases in bone, we hypothesized that osteoblasts might be a source of androgen synthesis. RNA expression analyses of murine osteoblasts and human bone confirmed that at least one 3βHSD and 17βHSD enzyme isoform was expressed, suggesting that osteoblasts are capable of generating androgens from adrenal DHEA. Murine osteoblasts were treated with 100 nM and 1 μM DHEA or vehicle control. Conditioned media from these osteoblasts were assayed for intermediate and active androgens by liquid chromatography-tandem mass spectrometry. As DHEA was consumed, the androgen intermediates androstenediol and androstenedione were generated and subsequently converted to T. Conditioned media of DHEA-treated osteoblasts increased androgen receptor (AR) signaling, prostate-specific antigen (PSA) production, and cell numbers of the androgen-sensitive prostate cancer cell lines C4-2B and LNCaP. DHEA did not induce AR signaling in osteoblasts despite AR expression in this cell type. We describe an unreported function of osteoblasts as a source of T that is especially relevant during androgen-responsive metastatic prostate cancer invasion into bone. © 2021 American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Histone demethylase KDM7A controls androgen receptor activity and tumor growth in prostate cancer.

Prostate cancer can be controlled by androgen-hormone treatment until the cancer becomes refractory. It is believed that hormone sensitivity is largely dependent on androgen receptor (AR) activity. Here, we found the histone demethylase KDM7A which demethylates histone H3K27 to be overexpressed in enzalutamide resistant castration-resistant prostate cancer cell line C4-2b, and investigated the molecular mechanism whereby androgen receptor activity is regulated by KDM7A. We engineered AR-positive LNCaP cells to stably express a short-hairpin RNA against KDM7A mRNA from a lentiviral vector. By measuring AR downstream gene expression after androgen stimulation, we found that a KDM7A-deficient cell line showed lower AR downstream gene expression compared to a control cell. KDM7A knock-down in LNCaP cell line caused decreased cell proliferation. Western blot analysis with modified-histone antibody revealed that the KDM7A-knock-down LNCaP cell line had increased H3K27 di-methylation. We confirmed KDM7A binding on AR target-gene promoters after hormone stimulation in chromatin-immunoprecipitation experiments. And increased H3K27 di-methylation was observed in KDM7A knock-down LNCaP stable cell. Treatment with KDM7A inhibitor, TC-E 5002, reduced proliferation and induced apoptosis of prostate cancer cells. Finally, we observed that the KDM7A protein was significantly upregulated in prostate cancer tissue, and that this difference correlated with the Gleason score. These data suggested that KDM7A is potentially a good therapeutic target for prostate cancer drugs and can be used as potentially a good prognostic indicator for prostate cancer and related treatment strategies.

MiR-543 Promotes Proliferation and Epithelial-Mesenchymal Transition in Prostate Cancer via Targeting RKIP

Background/Aims: MicroRNAs (miRNAs, miRs) have emerged as important post-transcriptional regulators in various cancers. miR-543 has been reported to play critical roles in hepatocellular carcinoma and colorectal cancer, however, the role of miR-543 in the pathogenesis of prostate cancer has not been fully understood. Methods: Expression of miR-543 and Raf Kinase Inhibitory Protein (RKIP) in clinical prostate cancer specimens, two prostate cancer cell lines, namely LNCAP and C4-2B, were determined. The effects of miR-543 on proliferation and metastasis of tumor cells were also investigated with both in vitro and in vivo studies. Results: miR-543 was found to be negatively correlated with RKIP expression in clinical tumor samples and was significantly upregulated in metastatic prostate cancer cell line C4-2B compared with parental LNCAP cells. Further studies identified RKIP as a direct target of miR-543. Overexpression of miR-543 downregulated RKIP expression and promoted the proliferation and metastasis of cancer cells, whereas knockdown of miR-543 increased expression of RKIP and suppressed the proliferation and metastasis of cancer cells in vitro and in vivo. Conclusion: Our study demonstrates that miR-543 promotes the proliferation and metastasis of prostate cancer via targeting RKIP.

Extracellular pH Modulates Neuroendocrine Prostate Cancer Cell Metabolism and Susceptibility to the Mitochondrial Inhibitor Niclosamide.

Neuroendocrine prostate cancer is a lethal variant of prostate cancer that is associated with castrate-resistant growth, metastasis, and mortality. The tumor environment of neuroendocrine prostate cancer is heterogeneous and characterized by hypoxia, necrosis, and numerous mitoses. Although acidic extracellular pH has been implicated in aggressive cancer features including metastasis and therapeutic resistance, its role in neuroendocrine prostate cancer physiology and metabolism has not yet been explored. We used the well-characterized PNEC cell line as a model to establish the effects of extracellular pH (pH 6.5, 7.4, and 8.5) on neuroendocrine prostate cancer cell metabolism. We discovered that alkalinization of extracellular pH converted cellular metabolism to a nutrient consumption-dependent state that was susceptible to glucose deprivation, glutamine deprivation, and 2-deoxyglucose (2-DG) mediated inhibition of glycolysis. Conversely, acidic pH shifted cellular metabolism toward an oxidative phosphorylation (OXPHOS)-dependent state that was susceptible to OXPHOS inhibition. Based upon this mechanistic knowledge of pH-dependent metabolism, we identified that the FDA-approved anti-helminthic niclosamide depolarized mitochondrial potential and depleted ATP levels in PNEC cells whose effects were enhanced in acidic pH. To further establish relevance of these findings, we tested the effects of extracellular pH on susceptibility to nutrient deprivation and OXPHOS inhibition in a cohort of castrate-resistant prostate cancer cell lines C4-2B, PC-3, and PC-3M. We discovered similar pH-dependent toxicity profiles among all cell lines with these treatments. These findings underscore a potential importance to acidic extracellular pH in the modulation of cell metabolism in tumors and development of an emerging paradigm that exploits the synergy of environment and therapeutic efficacy in cancer.

Abstract 1572: Overexpression of Axl delays metastatic outgrowth of prostate cancer bone marrow disseminated tumor cells

Abstract The presence of detectable metastases in the bone marrow of prostate cancer patients represents lethal, incurable disease. These metastases that may emerge years or decades after prostatectomy are derived from small colonies of dormant tumor cells that disseminated from the primary tumor. Our group has previously shown that Gas6/Axl signaling is associated with disseminated tumor cell (DTC) dormancy in mouse models of prostate cancer metastasis. In this study we explored the functional significance of this association and tested the hypothesis that overexpression of Axl in prostate cancer cells would prolong their dormant phase in the bone marrow. Axl overexpression in the AR-positive Axl-negative human prostate cancer cell line C42B slowed proliferation in vitro. Select cells were completely dormant as detected by the lack of EdU incorporation over 40 hours, and this fraction was increased when co-cultured with bone-forming osteoblasts. To assess the functional effects of Axl on prostate cancer progression in vivo, we utilized a mouse model of prostate cancer metastasis in which subcutaneous tumors are established and surgically removed upon reaching 200mm3. At 10 weeks post-tumor removal, mice bearing C42B-Axl tumors did not have overt osteoblastic or osteolytic lesions in their femurs or tibiae, whereas mice bearing C42B-WT tumors had visible lesions by x-ray. Immunohistochemistry staining of mouse femurs and tibiae corroborated these findings. To confirm that the absence of metastases in mice with C42B-Axl tumors was not due to the inability of the cells to disseminate, we used immunofluorescence to detect human cells in the bone marrow of flushed femurs and tibiae. At 12 weeks post-tumor removal, fewer prostate cancer cells were detected in the bone marrow of mice with C42B-Axl tumors compared to those with C42B-WT tumors. Notably however, both groups had detectable prostate cancer cells in the bone marrow, indicating that the absence of C42B-Axl metastasis was not due to failure of primary tumor cells to disseminate. The presence of disseminated C42B-Axl cells in the bone marrow coupled with their failure to develop into clinical metastases at the time of C42B-WT metastatic outgrowth establishes a functional role for the association of Axl expression with DTC dormancy. Axl may be sufficient to induce and maintain dormancy, and ongoing experiments will address the requirement of Axl in this setting. Citation Format: Haley D. Axelrod, Kenneth C. Valkenburg, Kenneth J. Pienta. Overexpression of Axl delays metastatic outgrowth of prostate cancer bone marrow disseminated tumor cells. [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 1572.

Abstract 749: Betaglycan-mediated regulation of mesenchymal stromal cell behavior in the prostate tumor-bone microenvironment

Abstract Prostate cancer frequently metastasizes to bone, resulting in increased risk of fractures and severe pain that significantly impacts patient quality of life. Bone metastatic prostate cancer is currently incurable with standard of care therapies being mainly palliative. In bone, prostate cancer generates extensive osteogenic lesions by promoting osteoblast activity. Osteoblasts are specialized bone forming cells derived from mesenchymal stem cells. In a bid to find new molecular targets through which prostate cancer induces osteogenesis, we incubated naïve MSCs with osteogenic bone metastatic prostate cancer cell line C4-2B conditioned media. Gene expression analysis revealed a 3-fold increase in expression of the TGFâ co-receptor, betaglycan. Given the critical role of TGFâ in the context of bone metastatic disease, we examined the role of betaglycan in MSC behavior. To this end, we utilized shRNA to knockdown betaglycan gene expression in mouse bone marrow-derived primary MSCs. Reduced betaglycan expression in knockdown (KD) MSCs had little to no impact on the expression of TGFâ signaling receptors, TGFâRI and TGFâRII, or pan-TGFâ ligand expression. However, using PAI promoter activity, a readout for TGFâ signaling, we observed a 3-fold increase in luminescence in KD-MSCs compared to controls. Likewise, phosphorylated Smad 2 was significantly increased in Betaglycan KD-MSCs in response to TGFâ treatment, collectively demonstrating increased TGFâ signaling in MSCs with reduced betaglycan expression. Using modified Boyden chamber assays, we found that 50% more Betaglycan-KD MSCs migrated towards recombinant TGFâ than controls (100 KD-MSCs cells migrated towards recombinant TGFâ vs. 50 control cells); this change was reversed with the addition of recombinant betaglycan. Using osteogenic differentiation assay, we found that Betaglycan KD-MSC differentiation was reduced by approximately 70% compared to control MSCs as measured by alizarin red staining. Addition of recombinant TGFâ further suppressed differentiation in both KD-MSCs and Control MSCs. This phenomenon was rescued by the addition of recombinant betaglycan, suggesting that MSC-derived betaglycan is critical for driving the osteogenic program. We are currently determining the impact of betaglycan on MSC behavior in the tumor bone microenvironment using in vivo models of bone metastatic prostate cancer. Collectively, these findings suggest that prostate cancer-induced MSC osteogenic programs are regulated in part via the induction of betaglycan. Citation Format: Leah M. Cook, Jeremy J. McGuire, Conor C. Lynch. Betaglycan-mediated regulation of mesenchymal stromal cell behavior in the prostate tumor-bone microenvironment. [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 749.

Abstract A35: Axl overexpression in a prostate cancer mouse model delays metastasis and may prolong dormancy of disseminated tumor cells

Abstract The most common site of metastasis in prostate cancer (PCa) patients is the bone marrow, where disseminated tumor cells (DTCs) preferentially home to the hematopoietic stem cell niche and can undergo a state of dormancy. These PCa cells may lie dormant in the bone marrow for years after prostatectomy, but may eventually re-enter the cell cycle resulting in lethal metastatic disease. Using mouse models of PCa metastasis, our group has previously shown that Gas6/Axl signaling is associated with dormant bone marrow DTCs. In this study we investigated the requirement of Axl in initiating and maintaining the dormant phase. We hypothesized that overexpression of Axl in PCa cells would prolong dormancy. Overexpression of Axl in the bone-homing AR-positive Axl-negative human PCa cell line C42B slows proliferation in vitro. Subcutaneous injection of C42B-Axl cells in a xenograft mouse model resulted in delayed onset of tumor growth relative to WT controls. C42B-WT tumors reached an average volume of 200mm3 at 6.6 weeks, while C42B-Axl tumors reached the same volume at 11 weeks. Regions of the primary tumor with a high percentage of Axl+ cells correlated with a low number of Ki67+ cells, indicating that Axl may inhibit proliferation even in the primary tumor. Surprisingly, C42B-Axl tumors were more vascular as observed by eye, and regions of increased vasculature correlated with a high percentage of Axl+ cancer cells. Metastatic outgrowth in soft tissues reached a lethal burden ~12 weeks following C42B-WT tumor removal, while mice bearing C42B-Axl tumors did not have detectable metastases by eye at the same timepoint post-tumor removal and were apparently healthy. Metastatic outgrowth in the bone was detected by the presence of osteoblastic and osteolytic activity at 5-12 weeks post-tumor removal in the femurs and tibiae of mice bearing C42B-WT tumors. At 8-12 weeks after C42B-Axl tumor removal, however, there was no evidence of metastatic bone disease. While these experiments are still ongoing, we have shown that Axl expression in the primary tumor delayed metastatic burden in both soft tissues and in bone in a mouse model of PCa metastasis. In ongoing studies, we will compare DTC number and time to metastasis in the bone marrow of C42B-WT and C42B-Axl tumor-bearing mice. Citation Format: Haley D. Axelrod, Kenneth C. Valkenburg, Kenneth J. Pienta. Axl overexpression in a prostate cancer mouse model delays metastasis and may prolong dormancy of disseminated tumor cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A35.

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

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

Different biological characteristics of different prostatic zonal primary stromal cells and cancer associated stromal cells,as well as their different effects on prostate cancer cell line C4-2B

Objective To compare the different biological characteristics of normal prostatic peripheral zonal primary stromal cells (normal prostatic peripheral fibroblasts,NPPF),transition zonal primary stromal cells (normal prostatic transitional fibroblasts,NPTF) and prostate cancer associated primary stromal cells (cancer associated fibroblasts,CAF),as well as their different effects on prostate cancer cell line C4-2B.Methods HE staining was done to identify the different histological characteristics of prostatic different zonal tissue and cancer tissue.NPPF,NPTF and CAF were primarily cultured,and the expression of Vimentin,smooth muscle actin (SMA) and prostatic specific antigen (PSA) was detected by using immunocytochemistry.Their proliferation was compared by growth curves,apoptosis rate by flow cytometry (FCM),and cellular ultrastructure by transmission electron microscopy.The co-culture system of primary stromal cells and C4-2B cells were established,and the changes in proliferation and apoptosis of C4-2B cells affected by different stromal cell subsets were compared.Results There was significant difference in proliferation,apoptosis,ultrastructure among NPPF,NPTF and CAF,and their biological effects on C4-2B cells.Growth rate of NPPF,NPTF and CAF was increased in turn,but their apoptosis decreased in turn [9.25 ± 2.24)%,(5.98 ± 0.74)%,(2.63 ± 0.96)%,respectively].Transmission electron microscopy suggested that CAF had the most productive protein synthesis.After C4-2B cells were co-cultured with the prostate stromal cell subsets,proliferation was increased,but apoptosis was decreased.Methylthiazol tetrazolium (MTT) assay showed that the absorbance in C4-2B cells after co-culture with NPPF,NPTF and CAF for 2 and 4 days was 0.540,0.471,0.632,and 0.554,0.488,0.670,respectively,P ＜0.05.FCM revealed that as compared with the control group (10.32 ±0.43)%,CAF had the strongest capability to inhibit the apoptosis of C4-2B cells (3.36±0.17)%,followed by NPPF (5.97 ±0.70)% and NPTF (8.01 ±0.22)%.Conclusion There are significantly different biological characteristics among NPPF,NPTF,CAF,which may be the main reason leading to the genesis and development of bengin prostatic hyperplasia or prostate cancer. Key words: Prostate cancer; Tansitional zone; Stromal cell; Coculture

Abstract 1490: Overexpression of a calcium channel protein TRPM8 in prostate cancer cells enhances their anchorage-independent growth and resistance to paclitaxel-induced apoptosis

Abstract The non-voltaged-gated transient receptor potential protein melastatin member 8 (TRPM8) has been characterized to function as a cold- and menthol-activated calcium-permeable channel protein, which it was originally cloned in a prostate-specific substracted cDNA library and shown to be up-regulated in benign prostatic hyperplasia and prostate cancer as compared to normal prostatic epithelial cells. Recent studies in cultured prostate cancer cells and clinical prostate cancer tissues show that TRPM8 is an androgen receptor-regulated calcium channel which is required for the survival and androgen-dependent growth of prostate cancer cells. Based on this evidence, it is believed that TRPM8 may play an important role in the advanced growth of prostate cancer. However, the prostate-specific function of TRPM8 and the role of TRPM8-regulated calcium-influx in prostate function and its carcinogenesis remain unclear. Our preliminary studies showed that TRPM8 was significantly over-expressed in an androgen-independent and metastatic prostate cancer cell line C4-2B as compared to its parental line LNCaP and also in two animal models of metastatic prostate cancers (a transgenic mouse model of prostate cancer-TGMAP and xenograft model of C4-2B). In order to elucidate the functional significance of TRPM8 in prostate cancer cell growth, we over-expressed TRPM8 in an androgen-sensitive prostate cancer cell line LNCaP by lentiviral transduction and generated stable TRPM8 clones for phenotypic growth studies. Our findings showed that stable ectopic TRPM8 expression induced morphological changes in LNCaP cells with reduction of filopodial processes and also suppressed their in vitro cell proliferation as shown by MTT assay. However, stable LNCaP-TRPM8 clones demonstrated significantly enhanced colony formation efficiencies in soft agar assay. Moreover, stable TRPM8 expression also enhanced the in vivo tumor growth of LNCaP cells in immunodeficient SCID mice and resistance to paclitaxel-induced apoptosis, as compared to their parental line. The results of our present study suggest that altered calcium-homeostasis as disturbed by TRPM8 overexpression in prostate cancer cells could enhance their in vitro anchorage-independent and in vivo tumor growth, and also resistance to paclitaxel-induced apoptosis, while the involved calcium-dependent signaling pathways still remain to be elucidated. (Study supported by a RGC Competitive Earmarked Research Grant project code 461007) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1490.

Calcium/calmodulin‐dependent kinase II regulates notch‐1 signaling in prostate cancer cells

Notch signaling is associated with prostate osteoblastic bone metastases and calcium/calmodulin-dependent kinase II (CaMKII) is associated with osteoblastogenesis of human mesenchymal stem cells. Here we show that prostate cancer cell lines C4-2B and PC3, both derived from bone metastases and express Notch-1, have all four isoforms of CaMKII (alpha, beta, gamma, delta). In contrast, prostate cancer cell lines LNcaP and DU145, which are not derived from bone metastases and lack the Notch-1 receptor, both lack the alpha isoform of CaMKII. In addition, DU145 cells also lack the beta-isoform. In C4-2B cells, inhibition of CaMKII by KN93 or gamma-secretase by L-685,458 inhibited the formation of the cleaved form of Notch-1 thus inhibiting Notch signaling. KN93 inhibited down stream Notch-1 signaling including Hes-1 gene expression, Hes-1 promoter activity, and c-Myc expression. In addition, both KN93 and L-685,458 inhibited proliferation and Matrigel invasion by C4-2B cells. The activity of gamma-secretase was unaffected by KN93 but markedly inhibited by L-685,458. Inhibition of the expression of alpha, beta, or gamma-isoform by siRNA did not affect Hes-1 gene expression, however when expression of one isoform was inhibited by siRNA, there were compensatory changes in the expression of the other isoforms. Over-expression of CaMKII-alpha increased Hes-1 expression, consistent with Notch-1 signaling being at least partially dependent upon CaMKII. This unique crosstalk between CaMKII and Notch-1 pathways provides new insight into Notch signaling and potentially provides new targets for pharmacotherapeutics.

Amplification and overexpression of prosaposin in prostate cancer

We identified prosaposin (PSAP) as a secreted protein expressed in androgen-independent (AI) prostate cancer cells by cloning/sequencing, after probing a PC-3 cDNA library expressed in the lambdaTriplEx phagemid expression vector with a polyclonal rabbit antibody generated against pooled human seminal plasma. PSAP is a neurotrophic molecule; its deficiency or inactivation has proved to be lethal in man and mice, and in mice, it leads to abnormal development and atrophy of the prostate gland, despite normal testosterone levels. We used Southern hybridization, quantitative real-time polymerase chain reaction, and/or single nucleotide polymorphism (SNP) array analysis, and we now report the genomic amplification of PSAP in the metastatic AI prostate cancer cell lines, PC-3, DU-145, MDA-PCa 2b, M-12, and NCI-H660. In addition, by using SNP arrays and a set of 25 punch biopsy samples of human prostate cancer xenografts (LAPC3, LuCaP 23.1, 35, 49, 58, 73, 77, 81, 86.2, 92.1, 93, 96, 105, and 115), lymph nodes, and visceral-organ metastases, we detected amplification of the PSAP locus (10q22.1) in LuCaP 58 and 96 xenografts and two lymph node metastases. In addition, AI metastatic prostate cancer cell lines C4-2B and IA8-ARCaP over-expressed PSAP mRNA without evidence of genomic amplification. Taken together with prior data that demonstrated the growth-, migration-, and invasion-promoting activities, the activation of multiple signal transduction pathways, and the antiapoptotic effect of PSAP (or one of its active domains, saposin C) in prostate cancer cells, our current observation of PSAP amplification or overexpression in prostate cancer suggests, for the first time, a role for this molecule in the process of carcinogenesis or cancer progression in the prostate.