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(A) LNCaP cells {plus minus} AD were treated with edelfosine (10 µM) and the cells were harvested after 3, 6 and 18 h time points. Cell lysates were examined by Western blotting for p-AKT and total AKT. (B) ARv7 expression in 22RV1 cells. Cells were cultured in 10 cm tissue culture dishes in CM or AD medium and then treated edelfosine (10 µM). Treated cells were harvested after 24 h, whole-cell lysates were extracted, and ARv7 expression was determined by Western immunoblotting. The same blot was re-probed for β-actin.

Supplementary Figures 1-3

Cells were harvested after 24 hours following treatment and caspase 3/7 assay was carried out. For ATF3 knockdown, VCaP were transfected with control siRNA or siATF3 (10 nM), followed by treatment with edelfosine (5 µM). Data shown represent the mean values ({plus minus} SEM) from three independent experiments. *p < 0.05, compared to control siRNA + edelfosine (one way ANOVA).

Androgen deprived LNCaP cells were co-transfected with 5 µg of ARE-luciferase reporter constructs, renilla luciferase and siATF3 (10 nM), followed by treatment with edelfosine (5 µM) and R1881 (5 nM). Cells were harvested and lysed after 24 hours following treatment and assayed for ARE reporter activity. Luciferase expression of each sample was measured using dual luciferase reporter gene assay system (Promega Corp., Madison, WI). The data represents the ratio between firefly/renilla luciferase activities. The mean values {plus minus} SEM of three independent experiments are shown. *p < 0.05, compared to group with R1881 alone (one way ANOVA).

S1. VSV-hIFNβ treatment followed by RT caused S-phase cell cycle arrest of prostate cancer cells. S2. VSV treatment in combination with radiation therapy increases the GFP or Luciferase expression in vitro and in vivo. S3. C57BL/6 CD8 Re-challenge study with VSV-mIFNβ+RT followed by CD8 depletion.

VCaP cells were grown for 24 h in complete (A) or AD (B) medium. Cells were treated with edelfosine (1, 2.5, 5, 10 and 20 µM) with control groups receiving PBS (0.01 M) vehicle (S1A) VCaP and (S1B) VCaP + AD. Cells were monitored and cell proliferation was measured in real time by RTCA cell proliferation assay and the average cell index was shown for 72 h. Each data point is represented by its means (n = 3) {plus minus} SD.

Supplementary Figure legends for Figures S1-S5.

Background. Historically, intraductal carcinoma of the prostate (IDC-P) was postulated to be a retrograde spread of high-grade invasive prostate cancer. There is evidence that IDC-P can primarily originate in the prostatic ducts. The retrograde genesis has never been experimentally or clinically confirmed before. Methods. Biopsy proven intermediate or high-risk prostate cancer was orthotopically grafted in the prostate of severe combined immunodeficiency gamma mice. Cancer growth was monitored by serum PSA levels. The animals were sacrificed and grafted areas were histological examined. Results. Twenty-one of 23 mice survived and demonstrated rising serum PSA. In 10 of 21 animals, human prostate cancer was identified orthotopically. Except for one case where the human biopsy showed a Grade Group 2 prostate cancer and mouse graft was Grade Group 5, other 9 specimens showed comparable grades. One of the specimens demonstrated a cribriform invasive prostate cancer and adjacent IDC-P. Conclusion. These experimental data offer some evidence that invasive prostate cancer can demonstrate a retrograde spread in the prostatic ducts as IDC-P. Its ability to primarily arise in the ducts has been demonstrated in other studies. However, the issue which remains unresolved is in its most common presentation of IDC-P intermixed with high-grade invasive cancer if it is the former or the latter which came first. Possibly resolving this dilemma will shed some light on the existing controversies if IDC-P should or should not be graded when invasive cancer is present.

Purpose: To investigate the relationships between circulating tumor cells (CTCs), free circulating DNA (fcDNA) and biochemical response in prostate cancer patients treated primarily versus salvage radiotherapy (RT). Methods and Materials: Blood was collected prospectively from patients, enrolled in two institutional Phase II trials for primary and salvage RT. Three blood samples were collected at: (i) prior to treatment [RT or androgen deprivation therapy (ADT)], (ii) last week of RT, and (iii) three months post-RT. CTCs were quantified in 31 samples from 12 primary patients and 30 samples from 12 salvage patients; fcDNA were analyzed in 11 primary (28 samples) and 5 (9 samples) salvage patients. CTCs were visualized by immunofluorescence after microfilter capture and fcDNA was quantified using real-time Polymerase chain reaction (PCR). CTCs and fcDNA were correlated with early biochemical response by subdividing patients into early favorable and unfavorable response at 3 months after RT. Results: For those treated primarily, there was a direct correlation with CTC counts and prostate specific antigen (PSA) pre-RT that changed to a reciprocal relationship 3 months post-RT. CTCs increased significantly (p=0.03) at 3 months after primary RT in the biochemical favorable patients, while no significant association was observed for fcDNA. Correspondingly, post-RT fcDNA levels were inversely related to CTC counts. In salvage patients, the number of CTCs was related to pre-RT PSA, but it was not correlated to RT response. In post-RT series, a significant direct correlation was observed between CTCs and PSA. Conclusion: Our preliminary studies suggest that RT affects CTC counts, which are thus associated with prostate cancer biochemical response. A larger cohort with longer follow-up will be needed to establish the association with more recognized treatment endpoints.

Purpose Small animal irradiation is crucial to the investigation of radiobiological mechanisms. The paradigm of clinical radiation therapy is trending toward high-precision, stereotactic treatment. However, translating this scheme to small animal irradiation is challenging owing to the lack of high-quality image guidance. To overcome this obstacle, we developed a multimodality image guided precision radiation platform. Methods and Materials The platform consists of 4 modules: x-ray computed tomography (CT), bioluminescence tomography (BLT), fluorescence molecular tomography (FMT), and radiation therapy. CT provides animal anatomy and material density for radiation dose calculation, as well as body contour for BLT and FMT reconstruction. BLT and FMT provide tumor localization to guide radiation beams and molecular activity to evaluate treatment outcome. Furthermore, we developed a Monte Carlo–based treatment planning system (TPS) for 3-dimensional dose calculation, calibrated it using radiochromic films sandwiched in a water-equivalent phantom, and validated it using in vivo dosimeters surgically implanted into euthanized mice (n = 4). Finally, we performed image guided irradiation on mice bearing orthotopic breast and prostate tumors and confirmed radiation delivery using γH2AX histology. Results The Monte Carlo–based TPS was successfully calibrated by benchmarking simulation dose against film measurement. For in vivo dosimetry measured in the euthanized mice, the average difference between the TPS calculated dose and measured dose was 3.86% ± 1.12%. Following the TPS-generated treatment plan, we successfully delivered 20 Gy dose to an animal bearing an orthotopic prostate tumor using 4 BLT-guided radiation beams and 5 Gy dose to an animal bearing an orthotopic breast tumor using a single FMT-guided radiation beam. γH2AX histology presented significantly more DNA damage in irradiated tumors and thus validated the dose delivery accuracy. Conclusions Combined with Monte Carlo TPS, this multimodality CT/BLT/FMT image guided small animal radiation platform can specifically localize tumors, accurately calculate dose distribution, precisely guide radiation delivery, and molecularly evaluate treatment response. It provides an advanced toolset for radiobiology and translational cancer research.

Multi-modality x-ray CT and optical molecular imaging with effective background subtraction enables specific and sensitive tumor detection, which is critical for precise small animal image-guided irradiation and early treatment response assessment.

Purpose X-ray CT faces challenges in differentiating tumors from surrounding healthy tissues. A bioluminescence tomography (BLT) system which can directly reconstruct the internal luminescent tumors, was developed and integrated with CT system to accurately guide radiation dose delivery. Methods The BLT system, employing a lens-coupled CCD camera, was physically registered with an onboard cone beam CT system in an image-guided small animal arc radiation treatment system (iSMAART). The onboard CT provides animal anatomy and accurate surface contour used to construct the three-dimensional mesh for the BLT reconstruction. Bioluminescence projections were captured from multiple angles, once every 90° rotation. The BLT reconstruction was performed on an orthotopic prostate tumor model to evaluate its robustness and accuracy in locating and delineating bioluminescent tumors. The location and volume of the tumor identified from iodinated contrast CT was used to validate the BLT performance. Phantom experiment was also conducted to confirm the precision of BLT-guided radiation. Results The BLT was able to accurately locate the bioluminescent tumors with < 0.5 mm error. The tumor volume in BLT was significantly correlated with that in the iodinated contrast CT (R2 = 0.81, P < 0.001). Phantom experiments further validated that BLT can be used to guide radiation with submillimeter accuracy. Conclusion Together with CT, BLT can provide precision radiation guidance and robust tumor volume assessment in small animal cancer research.

Vesicular stomatitis virus (VSV) expressing IFNβ induces apoptosis in multiple tumor models while maintaining an excellent safety profile. VSV-IFNβ is oncoselective due to permissive replication in cells with an altered IFN pathway. The human VSV-IFNβ (hIFNβ) vector is currently used in clinical trials as a standalone therapy; however, we hypothesized that oncolytic virotherapy might be more effective when used in combination with radiotherapy (RT). We investigated the synergistic effects of RT and VSV-hIFNβ in the subcutaneous PC3 and orthotopic LNCaP prostate xenograft models and a syngeneic RM9 prostate tumor model. VSV-IFNβ combined with RT amplified tumor killing for PC3 and LNCaP xenografts, and RM9 tumors. This was attributed to the induction of proapoptotic genes leading to increased VSV-IFNβ infection and replication, VSV expression, and oncolysis. In the RM9 tumors, combination therapy resulted in a robust antitumor immune response. Treated RM9 tumor-bearing mice demonstrated an increase in CD8+ and CD4+ T-cell numbers, 100% resistance to tumor rechallenge, and reduced resistance to reimplantation challenge with CD8+ knockdown. RT enhanced the activity of VSV-mediated oncolysis via attenuation of the innate antiviral response, resulting in increased VSV replication and the generation of an adaptive immune response earmarked by an increase in CD8+ lymphocyte numbers and antitumor activity. Local tumor irradiation combined with VSV-IFNβ affects tumor cell death through direct and systemic activity in conjunction with pronounced antitumor immunity. Implications: Radiotherapy enhances VSV-mediated oncolysis and anti-tumor immunity, indicating that the ombination has promise for very high risk prostate cancer.

Deregulation of microRNAs (miRs) contributes to progression and metastasis of prostate and other cancers. miR-23b and -27b, encoded in the same miR cluster (miR-23b/-27b), are downregulated in human metastatic prostate cancer compared with primary tumors and benign tissue. Expression of miR-23b/-27b decreases prostate cancer cell migration, invasion and results in anoikis resistance. Conversely, antagomiR-mediated miR-23b and -27b silencing produces the opposite result in a more indolent prostate cancer cell line. However, neither miR-23b/-27b expression or inhibition impacts prostate cancer cell proliferation suggesting that miR-23b/-27b selectively suppresses metastasis. To examine the effects of miR-23b/-27b on prostate cancer metastasis in vivo, orthotopic prostate xenografts were established using aggressive prostate cancer cells transduced with miR-23b/-27b or non-targeting control miRNA. Although primary tumor formation was similar between miR-23b/-27b-transduced cells and controls, miR-23b/-27b expression in prostate cancer cells decreased seminal vesicle invasion and distant metastases. Gene-expression profiling identified the endocytic adaptor, Huntingtin-interacting protein 1-related (HIP1R) as being downregulated by miR-23b/-27b. Increased HIP1R expression in prostate cancer cells inversely phenocopied the effects of miR-23b/-27b overexpression on migration, invasion and anchorage-independent growth. HIP1R rescued miR-23b/-27b-mediated repression of migration in prostate cancer cells. HIP1R mRNA levels were decreased in seminal vesicle tissue from mice bearing miR-23b/-27b-transduced prostate cancer cell xenografts compared with scrambled controls, suggesting HIP1R is a key functional target of miR-23b/-27b. In addition, depletion of HIP1R led to a more rounded, less mesenchymal-like cell morphology, consistent with decreased metastatic properties. Together, these data demonstrate that the miR-23b/-27b cluster functions as a metastasis-suppressor by decreasing HIP1R levels in pre-clinical models of prostate cancer.

Liposomes have been extensively studied and are used in the treatment of several diseases. Liposomes improve the therapeutic efficacy by enhancing drug absorption while avoiding or minimizing rapid degradation and side effects, prolonging the biological half-life and reducing toxicity. The unique feature of liposomes is that they are biocompatible and biodegradable lipids, and are inert and non-immunogenic. Liposomes can compartmentalize and solubilize both hydrophilic and hydrophobic materials. All these properties of liposomes and their flexibility for surface modification to add targeting moieties make liposomes more attractive candidates for use as drug delivery vehicles. There are many novel liposomal formulations that are in various stages of development, to enhance therapeutic effectiveness of new and established drugs that are in preclinical and clinical trials. Recent developments in multimodality imaging to better diagnose disease and monitor treatments embarked on using liposomes as diagnostic tool. Conjugating liposomes with different labeling probes enables precise localization of these liposomal formulations using various modalities such as PET, SPECT, and MRI. In this review, we will briefly review the clinical applications of liposomal formulation and their potential imaging properties.

The image guided small animal arc radiation treatment platform has adopted onboard cone beam computed tomography and bioluminescence tomography (BLT). We used BLT to guide irradiation delivery and quantitatively assess irradiation-induced tumor response.BLT was first validated on a tissue-simulating phantom, where the internal chemiluminescent liquid had a constant volume while its luminescence intensity gradually decayed. Then, in vivo experiments were performed on BALB/c mice orthotopically inoculated with 4T1 breast carcinoma cells expressing luciferase. Animals either received radiation treatment (radiation therapy [RT] group, n = 9) or did not (control group, n = 9). BLT was used to guide delivery of a single-fraction 5-Gy radiation dose to the tumor and to evaluate the treatment response. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining was used to evaluate irradiation-induced DNA damage and cell apoptosis.Phantom results showed that BLT not only recovered the constant target volume with2% deviation but also accurately monitored the decay of the chemiluminescent molecules. For the RT group of animals, there was significant reduction in both the BLT-based tumor volume (21% ± 10%, P = .001) and bioluminescence intensity (48% ± 17%, P = .0008). For the control group, a significant increase was detected in the BLT tumor volume (35% ± 12%, P .0001) but not the BLT bioluminescence intensity (P = .4). There was a significant difference in the BLT tumor volume between the RT and control groups 7 days after irradiation (P = .03). Regression analysis suggests a strong correlation between the BLT and cone beam computed tomography tumor volume (RBLT onboard the image guided small animal arc radiation treatment platform can be used to accurately guide irradiation delivery and to quantitatively assess treatment response by simultaneously monitoring tumor volume and cancer cell population.

Expression of carbonic anhydrase 9 (CA9) is associated with poor prognosis and increased tumor aggressiveness and does not always correlate with HIF-1α expression. Presently, we analyzed the regulation of CA9 expression during hypoxia by HIF-1α, Notch3, and the von Hippel-Lindau (VHL) in breast carcinoma cells. Both HIF-1α and Notch3 were absolutely required for the expression of CA9 mRNA, protein, and reporter. Reciprocal co-immunoprecipitation of HIF-1α, Notch3 intracellular domain (NICD3), and pVHL demonstrated their association. The presence of common consensus prolyl hydroxylation and pVHL binding motifs (L(XY)LAP);LLPLAP(2191) suggested an oxygen-dependent regulation for NICD3. However, unlike the HIF-1α protein, NICD3 protein levels were not modulated with hypoxia or hypoxia-mimetic agents. Surprisingly, mutations of the common prolyl hydroxylation and pVHL binding domain lead to the loss of CA9 mRNA, protein, and reporter activity. Chromatin immunoprecipitation assay demonstrated the association of NICD3, HIF-1α, and pVHL at the CA9 promoter. Further, the NICD3 mutant defective in prolyl hydroxylation and subsequent pVHL binding caused a reduction in cell proliferation of breast carcinoma cells. We show here for the first time that the interaction of HIF-1α with NICD3 is important for the regulation of CA9 expression. These findings suggest that although CA9 is a hypoxia-responsive gene, its expression is modulated by the interaction of HIF-1α, Notch3, and VHL proteins. Targeting the expression of CA9 by targeting upstream regulators could be useful in cancer/stem cell therapy.

Purpose Differentiating tumor from its surrounding soft tissues is challenging for x-ray computed tomography (CT). Fluorescence molecular tomography (FMT) can directly localize the internal tumors targeted with specific fluorescent probes. A FMT system was developed and integrated onto a CT guided irradiator to improve tumor localization for image guided radiation. Methods The FMT system was aligned orthogonal to the cone beam CT onboard our previously developed image guided small animal arc radiation treatment system (iSMAART). Through rigorous physical registration, the onboard CT provides accurate surface contour which is used to generate three-dimensional mesh for FMT reconstruction. During FMT experiments, a point laser source perpendicular to the rotating axis was used to excite the internal fluorophores. The normalized optical images from multiple projection angles were adopted for tomographic reconstruction. To investigate the accuracy of the FMT in locating the tumor and recovering its volume, in vivo experiments were conducted on two breast cancer models: MDA-MB-231 cancer xenograft on nude mice and 4T1 cancer xenograft on white mice. Both cancer cell lines overexpress the epidermal growth factor receptor (EGFR). A novel fluorescent poly(lactic-co-glycolic) acid (PLGA) nanoparticle conjugated with anti-EGFR was intravenously injected to specifically target the breast cancer cells. Another ex vivo experiment on a mouse bearing a surgically implanted Indocyanine Green-containing glass tube was conducted, to additionally validate the precision of FMT guided radiation therapy. Results The FMT can accurately localize the single-nodule breast tumors actively targeted with fluorescent nanoparticles with localization error

We have previously shown in separate studies that MDM2 knockdown via antisense MDM2 (AS-MDM2) and E2F1 overexpression via adenoviral-mediated E2F1 (Ad-E2F1) sensitized prostate cancer cells to radiation. Because E2F1 and MDM2 affect apoptosis through both common and independent pathways, we hypothesized that coupling these two treatments would result in increased killing of prostate cancer cells. In this study, the effect of Ad-E2F1 and AS-MDM2 in combination with radiation was investigated in three prostate cancer cell lines: LNCaP cells, LNCaP-Res cells [androgen insensitive with functional p53 and androgen receptor (AR)], and PC3 cells (androgen insensitive, p53null, and ARnull). A supra-additive radiosensitizing effect was observed in terms of clonogenic inhibition and induction of apoptosis (caspase-3 + caspase-7 activity) in response to Ad-E2F1 plus AS-MDM2 treatments in all three cell lines. In LNCaP and LNCaP-Res, these combination treatments elevated the levels of phospho-Ser15 p53 with significant induction of p21waf1/cip1, phospho-γH2AX, PUMA, and Bax levels and reduction of AR and bcl-2 expression. Similarly, ARnull and p53null PC-3 cells showed elevated levels of Bax and phospho-γH2AX expression. These findings show that the combination of Ad-E2F1 and AS-MDM2 significantly increases cell death in prostate cancer cells exposed to radiation and that this effect occurs in the presence or absence of AR and p53. (Mol Cancer Res 2008;6(11):1742–54)

Small animal radiotherapy studies should be performed preferably on irradiators capable of focal tumor irradiation and healthy tissue sparing. In this study, an image guided small animal arc radiation treatment system (iSMAART) was developed which can achieve highly precise radiation targeting through the utilization of onboard cone beam computed tomography (CBCT) guidance. The iSMAART employs a unique imaging and radiation geometry where animals are positioned upright. It consists of a stationary x-ray tube, a stationary flat panel detector, and a rotatable and translational animal stage. System performance was evaluated in regards to imaging, image guidance, animal positioning, and radiation targeting using phantoms and tumor bearing animals. The onboard CBCT achieved good signal, contrast, and sub-millimeter spatial resolution. The iodine contrast CBCT accurately delineated orthotopic prostate tumors. Animal positioning was evaluated with ~0.3 mm vertical displacement along superior-inferior direction. The overall targeting precision was within 0.4 mm. Stereotactic radiation beams conformal to tumor targets can be precisely delivered from multiple angles surrounding the animal. The iSMAART allows radiobiology labs to utilize an image guided precision radiation technique that can focally irradiate tumors while sparing healthy tissues at an affordable cost.

Edelfosine is a synthetic alkyl-lysophospholipid that possesses significant antitumor activity in several human tumor models. Here, we investigated the effects of edelfosine combined with androgen deprivation (AD) in LNCaP and VCaP human prostate cancer cells. This treatment regimen greatly decreased cell proliferation compared with single agent or AD alone, resulting in higher levels of apoptosis in LNCaP compared with VCaP cells. Edelfosine caused a dose-dependent decrease in AKT activity, but did not affect the expression of total AKT in either cell line. Furthermore, edelfosine treatment inhibited the expression of androgen receptor (AR) and was associated with an increase in activating transcription factor 3 (ATF3) expression levels, a stress response gene and a negative regulator of AR transactivation. ATF3 binds to AR after edelfosine + AD and represses the transcriptional activation of AR as demonstrated by PSA promoter studies. Knockdown of ATF3 using siRNA-ATF3 reversed the inhibition of PSA promoter activity, suggesting that the growth inhibition effect of edelfosine was ATF3 dependent. Moreover, expression of AR variant 7 (ARv7) and TMPRSS2-ERG fusion gene were greatly inhibited after combined treatment with AD and edelfosine in VCaP cells. In vivo experiments using an orthotopic LNCaP model confirmed the antitumor effects of edelfosine + AD over the individual treatments. A significant decrease in tumor volume and PSA levels was observed when edelfosine and AD were combined, compared with edelfosine alone. Edelfosine shows promise in combination with AD for the treatment of prostate cancer patients. Mol Cancer Ther; 15(6); 1353–63. ©2016 AACR.

Purpose: CT is not able to differentiate tumors from surrounding soft tissue. This study is to develop a bioluminescence tomography (BLT) system that is integrated onto our previously developed CT guided small animal arc radiation treatment system (iSMAART) to guide radiation, monitor tumor growth and evaluate therapeutic response. Methods: The BLT system employs a CCD camera coupled with a high speed lens, and is aligned orthogonally to the x-ray beam central axis. The two imaging modalities, CT and BLT, are physically registered through geometrical calibration. The CT anatomy provides an accurate contour of animal surface which is used to construct 3D mesh for BLT reconstruction. Bioluminescence projections are captured from multiple angles, once every 45 degree rotation. The diffusion equation based on analytical Kirchhoff approximation is adopted to model the photon propagation in tissues. A discrete cosine transform based reweighted L1-norm regularization (DCT-re-L1) algorithm is used for BLT reconstruction. Experiments are conducted on a mouse orthotopic prostate tumor model (n=12) to evaluate the BLT performance, in terms of its robustness and accuracy in locating and quantifying the bioluminescent tumor cells. Iodinated contrast agent was injected intravenously to delineate the tumor in CT. The tumor location and volume obtained from CT also serve as a benchmark against BLT. Results: With our cutting edge reconstruction algorithm, BLT is able to accurately reconstruct the orthotopic prostate tumors. The tumor center of mass in BLT is within 0.5 mm radial distance of that in CT. The tumor volume in BLT is significantly correlated with that in CT (R2 = 0.81). Conclusion: The BLT can differentiate, localize and quantify tumors. Together with CT, BLT will provide precision radiation guidance and reliable treatment assessment in preclinical cancer research.

Previously we have shown that the matrix metalloproteinase matrilysin (MMP-7) is overexpressed in human prostate cancers compared with normal epithelium. However, the mechanism for this overexpression is not understood. Human prostate fibroblasts have been shown to express certain fibroblast growth factors (FGFs), including FGF-1. Evidence from our laboratory and others has indicated that FGFs can regulate the expression of certain matrix metalloproteinases, including matrilysin. The goal of this study was to determine whether pharmacological inhibition of FGFR signaling would alter LNCaP tumor growth as well as expression of promatrilysin when LNCaP cells were co-injected subcutaneously with human prostate fibroblasts into athymic nude mice. For these inhibitor studies, AG1-X2 beads were coated with the pharmacological FGFR inhibitor SU5402 and were co-injected along with LNCaP and human prostate fibroblast cells (PF). Mice injected with LNCaP/PF and LNCaP/PF/beads alone demonstrated significant tumor growth, whereas mice injected with LNCaP/PF/SU5402-coated beads showed a significant decrease in tumor volume and weight. Immunohistochemical analysis showed that significant promatrilysin expression in tumors was inhibited by the FGFR inhibitor SU5402. Serum prostate-specific antigen (PSA) and promatrilysin levels were measured by enzyme-linked immunosorbent assay. The mice injected with LNCaP/PF and LNCaP/PF/beads expressed promatrilysin and serum PSA levels that were inhibited by co-injecting with SU5402. Therefore, pharmacological inhibition of FGF receptor signaling results in a decrease in the growth of LNCaP tumors generated subcutaneously by co-injecting LNCaP cells and human prostate fibroblasts. The inhibition in tumor growth was correlated with a decrease in tumor promatrilysin expression and a decrease in serum promatrilysin and PSA.

The MMP, matrilysin. (20MMP-7), has been shown to be overexpressed in prostate cancer cells and to increase prostate cancer cell invasion. Prostate stromal fibroblasts secrete factor(s), including fibroblast growth factor-1. (20FGF-1) that induces promatrilysin expression in LNCaP cells. In the present study, we investigated the signal transduction pathway involved in the FGF-1-induced expression of promatrilysin. FGF-1 treatment significantly increased the activation of extracellular signal-regulated kinases 1 and 2. (20ERK1 and ERK2). This induction was time-dependent and was sustained until 24 hours after treatment. Treating the cells with MEK1/2 inhibitor. (20PD98059) eliminated ERK activation completely and blocked FGF-1-mediated induction of promatrilysin expression. Transient transfection studies with human matrilysin promoter resulted in a four-to-five-fold increase in reporter luciferase enzyme activity that was blocked by the MEK1/2 inhibitor. (20PD98059). Serine phosphorylation of signal transducer and activator of transcription 3. (20STAT3) was observed after FGF-1 treatment and pretreatment with 20 µM PD98059 abolished STAT3 phosphorylation. Transient transfection with dominant negative STAT3 inhibited FGF-1-induced transactivation of the matrilysin promoter indicating that STAT3 plays an important role in FGF-1-induced matrilysin expression. We propose that the FGF-1-induced signaling pathway that leads to promatrilysin expression is ERK-dependent and leads to phosphorylation of Ser-727 on STAT3, phosphorylated STAT3, then binds and transactivates the matrilysin promoter. Our results demonstrate that ERK-MAP kinase and transcription factor STAT3 are important components of FGF-1-mediated signaling, which induce promatrilysin expression in LNCaP cells.

Background: The underlying mechanisms responsible for the renal failure and proteinuria in radiation nephropathy remain largely unknown. Radiotherapy with or without chemotherapy may result in radiation-induced kidney injury in pelvic malignancies such as gynecologic cancers, lymphomas, gastrointestinal cancers, sarcomas of the upper abdomen and during total body irradiation. The current study investigates the role of sphingolipids in radiation-induced podocytopathy using a murine model. The molecular and functional effects of kidney irradiation were evaluated after single-dose exposures. Material/Methods: In cell culture, SMPDL3b expression post radiation (8Gy) was determined by real-time PCR (RT-PCR) and Western blotting. Morphological changes and DNA damage were detected post radiation using immunofluorescence microscopy. Wild-type C57BL/6 male and female mice (age 10-14 weeks) were irradiated with a single dose of X-ray (14 Gy) using an image guided small animal arc radiation treatment system (iSMAART). Rituximab/IgG was administered (50 mg/kg, intraperitoneal injection) 30 min before the single dose of irradiation. Functional kidney parameters, kidney histology, and gene expression were analyzed at 20, 30 and 40 weeks after irradiation. Results: Following irradiation, SMPDL3b expression at protein level was significantly reduced in vitro and in vivo. However, no significant changes were observed at the transcriptional level. Podocyte number also decreased significantly post radiation in vivo. iSMAART dynamic contrast enhanced (DCE) imaging data analysis showed reduced glomerular filtration rate post radiation. In agreement with the functional data, hematoxylin and eosin staining of kidney sections showed a multifocal increase in the number of pericytes, tubular atrophy, and glomerular damage. Periodic Acid-Schiff (PAS) staining showed an increase in glomerular mesangial matrix accumulation post radiation. Sirius red staining showed diffuse intertubular fibrosis, especially in the renal cortex post radiation. These histological changes were paralleled by the change in serum Creatinine, urine albumin. Rituximab pretreatment to mice, improved kidney functional parameters, vascular structure, normalization of pericyte coverage, suppress the development of fibrosis and tubular damage post irradiation. Conclusion: This study shows that rituximab pretreatment protects mice against radiation-induced nephrotoxicity, which may have therapeutic implications for radiation-induced injuries in cancer patients. Citation Format: Anis Ahmad, Alla Mitrofanova, Saba Ansari, Thirupandiyur Udayakumar, Jacek Bielawski, Alan Pollack, Alesia Fornoni, Brian Marples, Youssef Zeidan. Podocyte-specific SMPDl3b modulates radiation-induced renal dysfunction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 904. doi:10.1158/1538-7445.AM2017-904

Androgen deprivation therapy (ADT) is a mainstay of treatment for high-risk non metastatic and metastatic prostate cancer. Patients initially respond to ADT, but eventually develop resistance to treatment. To overcome these limitations, we have used oncolytic virotherapy which has the potential to influence both local and systemic disease through direct cytotoxic and immune mechanisms. Here, we investigated the effectiveness of radiation therapy (RT) combined with Vesicular Stomatitis Virus (VSV) vector expressing interferon β (IFNβ) in VSV resistant prostate xenografts. In initial cell culture experiments, microarray analysis indicated that PC3 cells treated with VSV-hIFNβ and RT displayed several genes known to directly inhibit VSV replication (e.g., MX2, OAS2, TRIM25, IFITM1) were downregulated compared with VSV-hIFNβ therapy alone. Additionally, many apoptotic pathway-associated genes (e.g. IL24, GADD45B, KLF4&10, TNF, EGFR) were upregulated several fold during combination therapy. Gene expression changes were confirmed by RT-PCR. Further, immunoblot analysis demonstrated expression levels of IFNAR, BCL-XL, phosphorylated AKT, JAK2 and STAT3 were also markedly decreased when VSV-hIFNβ was combined with RT. However, RIG-I was not significantly altered after VSV-hIFNβ+RT treatment. A marked decrease in MAVS, NFKB (p50), IRF7 and antiviral protein PKR was seen after combined treatment. The role of IFN pathway in viral response was investigated using an interferon-inducible promoter ISRE-luciferase (ISRE-Luc) reporter construct. A significant difference (>600 fold) in the ISRE-Luc activation was seen for PC3 compared to LNCaP cells, indicating that the IFN pathway is highly active in PC3 cells after VSV infection alone. The activation of ISRE-mediated transcription of luciferase was significantly attenuated (p Citation Format: Thirupandiyur S. Udayakumar, Dillon Betancourt, Glen Barber, Anis Ahmad, Brian Marples, Alan Pollack. Reduction in prostate tumor growth and induction of anti-tumor immunity after combined treatment with oncolytic VSV and radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 824. doi:10.1158/1538-7445.AM2017-824

Matrix metalloproteinases (MMPs) degrade extracellular matrix proteins, and there is evidence that they play a role in tumor cell growth, invasion and metastasis. Matrilysin (MMP-7) is over-expressed in prostate cancer cells and increases prostate cancer cell invasion. Prostate stromal fibroblasts secrete a factor(s), including fibroblast growth factor-1 (FGF-1), which induces promatrilysin expression in the prostate carcinoma cell line LNCaP but not in normal prostate epithelial cells (PrECs). Since FGF-1 is present in the prostate, an altered sensitivity to FGF-1 might explain the up-regulation of matrilysin expression in prostate cancer cells compared to normal prostate epithelium. FGF receptor-1 (FGFR-1) is not normally expressed by normal prostate epithelial cells; however, aberrant expression of this receptor has been reported in prostate cancer cells, including the LNCaP cell line. We hypothesized that aberrant expression of FGFR-1 in PrECs would render them sensitive to induction of promatrilysin expression by recombinant FGF-1. To test this hypothesis, we transiently transfected PrECs with an FGFR-1 expression vector, which resulted in over-expression of FGFR-1 protein in approximately 40% of cells. FGF-1 increased promatrilysin expression in FGFR-1-transfected PrECs 4-fold over mock-transfected cells, and this induction was inhibited by a specific FGFR-1 inhibitor, SU5402, and by co-expression of a dominant negative FGFR-1 protein. Our results demonstrate that aberrant FGFR-1 expression, an epigenetic phenomenon that has been associated with prostate cancer progression, allows induction of promatrilysin expression by FGF-1 in PrECs.

Previously, our laboratory showed that interleukin-1beta (IL-1beta) secreted by lipopolysaccharide-activated monocytes induces promatrilysin expression in the prostate carcinoma cell line, LNCaP. We now demonstrate that IL-1beta-induced promatrilysin expression is mediated by an indirect mechanism that requires nuclear factor Kappa B (NFkappaB)-dependent synthesis of IL-6. Inhibition of protein synthesis with cycloheximide blocked IL-1beta-mediated induction of matrilysin mRNA suggesting that synthesis of one or more additional factors is required for IL-1beta-induced promatrilysin protein expression. Blockage of NFkappaB transactivation activity abrogated IL-1beta-induced promatrilysin expression to baseline levels suggesting that NFkappaB transactivation activity is necessary. Inhibition of IL-6 activity attenuated IL-1beta-induced promatrilysin, but not NFkappaB transactivation activity indicating that IL-6 acts downstream of NFkappaB in potentiation of IL-1beta-mediated promatrilysin expression. Inhibition of protein synthesis with cycloheximide did not alter IL-6-induced induction of matrilysin mRNA indicating that, contrary to the mechanism by which IL-1beta regulates promatrilysin expression, IL-6-mediated matrilysin mRNA expression does not require new protein synthesis. Transient transfection with dominant negative STAT3 inhibited IL-1beta- and IL-6-induced promatrilysin. These data provide evidence that NFkappaB-mediated IL-6 synthesis is required for IL-1beta-induced promatrilysin expression, and IL-6 signaling through STAT3 plays a role in IL-1beta-induced promatrilysin expression.

BACKGROUND It has been determined that prostate cancer cells overexpress the matrix metalloprotease matrilysin (MMP-7), but the factors regulating this expression have not been identified. Fibroblast growth factors (FGF), which are expressed in the prostate, might participate in paracrine regulation of matrilysin expression by prostate cancer cells. METHODS We tested the ability of recombinant FGF proteins and prostate fibroblast-conditioned media (PFCM) to induce promatrilysin expression in the prostate carcinoma cell line, LNCaP, and in normal prostate epithelial (PrEC) cells. We also characterized prostate fibroblast FGF expression by reverse transcriptase-polymerase chain reaction (RT-PCR). An inhibitor of FGF receptor activation (SU5402) was used to determine the role of FGF proteins in the induction of promatrilysin expression by PFCM. RESULTS Recombinant FGF-1, FGF-2, FGF-9, FGF-10, and PFCM significantly induced promatrilysin expression in LNCaP cells but not in PrEC cells. Prostate fibroblasts express mRNAs for these FGF proteins, and inhibition of LNCaP cell FGF receptors with SU5402 substantially reduced the induction of promatrilysin expression by PFCM. CONCLUSIONS Stromally expressed FGF proteins induce promatrilysin expression in a prostate carcinoma cell, and may provide a mechanism for the overexpression of promatrilysin observed in prostate cancer. Prostate 41:215–223, 1999. © 1999 Wiley-Liss, Inc.

Our original hypothesis was AKT regulates the RNA-induced silencing Complex (RISC) activity through the phosphorylation of AGO proteins in Prostate carcinoma cells . Objectives: To test the hypothesis following specific aims were proposed. Aim 1: To show that AKT regulates the miRNA expression profile in prostate carcinoma cells. Aim 2: To show that AKT mediated phosphorylation of AGO proteins regulates the activity of RISC complex. Aim 3: To show that abrogation of AKT mediated phosphorylation of AGO proteins sensitizes prostate carcinoma cells to chemotherapy. For Aim 1, we initially tried studying the interaction of Ago (pan) with 14-3-3. We could see interaction between Ago(pan) and (tau) isoform of 14-3-3. We treated prostate carcinoma cells (PC3) with 40 M AKT inhibitor V, Triciribine.Total RNA was isolated and miRNA and mRNA expression analysis in prostate carcinoma cells (PC3) was carried out to identify the miRNA-mRNA correlation matrices using Pearson Correlation in response to cells treated with AKT inhibitor (Triciribine). We also identified the miRNA-mRNA correlation matrices using Pearson Correlation in response to cells treated with AKT inhibitor (Triciribine)These matrices were further classified into four groups viz., I. miRNA up mRNA down. II miRNA down mRNA up. III. Both miRNA and mRNA up. IV. Both miRNA and mRNA down..Using the GeneGo analysis we identified TGF- dependent induction of Epithelial Mesenchymal Transition (EMT) via SMADs as its target. For aim 2, in order to identify the miRNA associated with specific AGO proteins, Immunoprecipitation was carried out.Since, the quality of miRNA isolated was poor, we could not proceed further on this aim. Once the miRNA-mRNA pair regulated by PI3K/AKT/14-3-3 is identified, we could then test the hypothesis that AKT regulates the RNA-induced silencing Complex (RISC) activity through the phosphorylation of KSRP protein in Prostate carcinoma cells .

Both the p53- and E2F1-signaling pathways are defective in almost all types of tumors, suggesting very important roles for their signaling networks in regulating the process of tumorigenesis and therapy response. Studies on Radiation Therapy Oncology Group tissue samples have identified aberrant expression of p53, MDM2 (an E3 ubiquitin ligase that targets p53 for proteosomal degradation), and p16 (an upstream regulator of retinoblastoma and hence E2F1 in prostate cancer); abnormal expression of these biomarkers has been associated with clinical outcome after radiotherapy ± androgen deprivation therapy. Although the proapoptotic properties of p53 are well documented, a relatively new aspect of p53 function as an active mediator of prosurvival signaling pathways is now emerging. E2F1 is a transcription factor that possesses both proapoptotic and prosurvival properties. Thus, the role of E2F1 in the process of tumorigenesis versus apoptosis is a contested issue that needs to be resolved. Furthermore, the role of E2F1 in DNA repair is being increasingly recognized. Thus, novel approaches to curb the prosurvival and DNA repair capability of E2F1 while promoting apoptotic function are of interest. In this review, we discuss the challenges involved in targeting the p53/E2F1 pathways and the crosstalk networks, and further propose potential therapeutic strategies for prostate cancer management.

Purpose: We previously showed that E2F1 overexpression radiosensitizes prostate cancer cells in vitro. Here, we demonstrate the radiosensitization efficacy of adenovirus (Ad)-E2F1 infection in growing (orthotopic) LNCaP and (subcutaneous) PC3 nude mice xenograft tumors. Methods and Materials: Ad-E2F1 was injected intratumorally in LNCaP (3 x 10{sup 8} plaque-forming units [PFU]) and PC3 (5 x 10{sup 8} PFU) tumors treated with or without radiation. LNCaP tumor volumes (TV) were measured by magnetic resonance imaging, caliper were used to measure PC3 tumors, and serum prostate-specific antigen (PSA) levels were determined by enzyme-linked immunosorbent assay. Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, and key proteins involved in cell death signaling were analyzed by Western blotting. Results: Intracellular overexpression of Ad-E2F1 had a significant effect on the regression of TV and reduction of PSA levels relative to that of adenoviral luciferase (Ad-Luc)-infected control. The in vivo regressing effect of Ad-E2F1 on LNCaP tumor growth was significant (PSA, 34 ng/ml; TV, 142 mm{sup 3}) compared to that of Ad-Luc control (PSA, 59 ng/ml; TV, 218 mm{sup 3}; p

We have previously shown in separate studies that MDM2 knockdown via antisense MDM2 (AS-MDM2) and E2F1 overexpression via adenoviral-mediated E2F1 (Ad-E2F1) sensitized prostate cancer cells to radiation. Because E2F1 and MDM2 affect apoptosis through both common and independent pathways, we hypothesized that coupling these two treatments would result in increased killing of prostate cancer cells. In this study, the effect of Ad-E2F1 and AS-MDM2 in combination with radiation was investigated in three prostate cancer cell lines: LNCaP cells, LNCaP-Res cells [androgen insensitive with functional p53 and androgen receptor (AR)], and PC3 cells (androgen insensitive, p53(null), and AR(null)). A supra-additive radiosensitizing effect was observed in terms of clonogenic inhibition and induction of apoptosis (caspase-3 + caspase-7 activity) in response to Ad-E2F1 plus AS-MDM2 treatments in all three cell lines. In LNCaP and LNCaP-Res, these combination treatments elevated the levels of phospho-Ser(15) p53 with significant induction of p21(waf1/cip1), phospho-gammaH2AX, PUMA, and Bax levels and reduction of AR and bcl-2 expression. Similarly, AR(null) and p53(null) PC-3 cells showed elevated levels of Bax and phospho-gammaH2AX expression. These findings show that the combination of Ad-E2F1 and AS-MDM2 significantly increases cell death in prostate cancer cells exposed to radiation and that this effect occurs in the presence or absence of AR and p53.

BACKGROUND We and others have shown that the matrix metalloproteinases, MT1-MMP is overexpressed in human prostate PIN lesions and invasive cancers compared to normal prostate epithelium. However, the mechanism for this overexpression is not understood. Evidence from our laboratory and others has indicated that fibroblast growth factors (FGFs) can regulate the expression of certain matrix metalloproteinase. In addition, human prostate fibroblasts are known to express certain FGFs, including FGF-1. The purpose of the work in this paper was to determine the mechanism involved in FGF-1 induced MT1-MMP expression in prostate carcinoma cells. METHODS We tested the ability of recombinant FGF-1 to induce MT1-MMP expression in prostate carcinoma cell line, LNCaP cells. We measured the MT1-MMP message by using Northern analyses and protein levels by Western analysis after FGF-1 treatment. Downstream signaling was investigated using dominant negative constructs for FGFR-1 and signal transducer and activator of transcription-3 (STAT3). Transient transfection was performed using reporter plasmids of the MT1-MMP gene promoter region (7.2 kb) linked to the firefly luciferase gene in the pGL3-Basic vector. For dominant negative studies FGFR-1 dominant negative plasmid in PCEP4 vector or STAT3 dominant negative plasmid in pCMV-1 vector was co-transfected with the MT1-MMP reporter plasmid. RESULTS Recombinant FGF-1 significantly induced MT1-MMP expression in LNCaP prostate carcinoma cells. MT1-MMP message increased with FGF-1 treatment compared to that of untreated control LNCaP cells. Quantitation by digital image analysis revealed that this increase was twofold over untreated LNCaP cells. Treatment of pGL3-MT1-MMP-luciferase transfected cells with FGF-1 resulted in a twofold to fourfold increase in luciferase enzyme activity compared with untreated cells. Co-transfection of LNCaP with human MT1-MMP reporter construct and a dominant negative FGFR1 mutant showed that FGF-1-induced MT1-MMP expression in LNCaP cells was completely inhibited by the mutated FGFR-1, indicating that FGF receptor (FGFR) activation is necessary for induction of MT1-MMP. Further, expression of dominant negative STAT3 inhibited the FGF-1-induced transactivation of the human MT1-MMP 7.2-kb promoter. CONCLUSIONS From these data, we conclude that FGF-1 induces MT1-MMP expression in prostate carcinoma cells through a transcriptional mechanism mediated through the FGFR and the transcription factor, STAT3. These results confirm earlier data indicating that acidic FGF and STAT3 are involved in the signaling leading to the expression of a MMP. Our findings support the idea that paracrine and autocrine factors play an important role in the regulation of MT1-MMP in human prostate carcinoma cells. © 2003 Wiley-Liss, Inc.

Degradation of the extracellular matrix by proteolytic enzymes is a central aspect of physiological and pathologic tissue-remodeling processes such as trophoblastic implantation, wound healing, and tumor invasion. We have hypothesized that prostate adenocarcinoma cell invasion through the normal basal lamina is attributable in part to metalloproteinase-induced cleavage of laminin-5 (Ln-5) and enhanced motility of the cancer cells. We studied the role of membrane type-1-matrix metalloproteinase (MT1-MMP) expressed on the surface of prostate tumor cells in cleaving Ln-5 and enhancing the migration of prostate tumor cells. We also determined the nature of the MT1-MMP cleavage of human Ln-5 and how this altered Ln-5 changes the migration of prostate carcinoma cells. We found that human MT1-MMP cleaves purified human Ln-5 to an 80-kDa fragment. Mass spectrometry analyses of the 80-kDa cleaved product by trypsin and chymotrypsin gave 14 and 9 different peptide sequences, respectively, that were identical to the expected amino acid sequence of the Ln-5-beta3 chain. The recovered peptides represent 14.4% (trypsin) and 10.3% (chymotrypsin) of Ln-5-beta3 chain by amino acid count. Both trypsin and chymotrypsin digestion of MT1-MMP-cleaved product of Ln-5 did not show any other peptides that were identical to the other chains of Ln-5. Using a linear migration assay we found that the Ln-5 cleaved by MT1-MMP enhanced the migration of DU-145 prostate carcinoma cells by 2-fold compared with uncleaved Ln-5. The use of blocked antisense MT1-MMP oligonucleotides inhibited the migration of DU-145 cells on Ln-5. We also found that the prostate carcinoma cells expressing high levels of MT1-MMP, such as PC3N and PPC, demonstrated enhanced migration on human Ln-5-coated substrate, and this migration was inhibited using blocked antisense MT1-MMP oligonucleotides. In conclusion, this is a novel and important finding where we have shown that beta3-chain is cleaved by MT1-MMP, and this cleavage enhances migration of prostate cancer cells.

BACKGROUND We have shown previously that interleukin (IL) -1β– and IL-6–induced promatrilysin expression is mediated by an indirect pathway that requires NFκB-dependent synthesis of IL-6 and STAT3 signaling. We now demonstrate that IL-1β–induced but not IL-6–induced promatrilysin expression can be blocked by androgens in the prostate carcinoma cell line LNCaP (lymph node–derived carcinoma cells of the prostate). METHODS By using enzyme-linked immunosorbent assay analyses, promatrilysin was measured in LNCaP cells stimulated with IL-1β or IL-6 LNCaP-treated cells pretreated with testosterone. In addition, promatrilysin message was measured by using Northern analyses after IL-6–treated cells pretreated with testosterone. RESULTS In LNCaP treated with testosterone before IL-1β stimulation induced promatrilysin expression was completely abrogated. Furthermore, testosterone completely abrogated NFκB transactivation activity and induction of IL-6 protein expression and mRNA. Testosterone and 5α-dihydrotestosterone did not have an inhibitory effect on IL-6–induced promatrilysin expression. Testosterone also had no effect on basal promatrilysin expression or basal NFκB transactivation activity. CONCLUSION From these data, we conclude that testosterone blocks IL-1β–induced promatrilysin expression by inhibition of NFκB transactivation activity, which in turn, blocks IL-6 expression. These data suggest a mechanism in vivo by which invasive and metastatic prostatic carcinoma cell clones refractory to hormone ablation therapy may develop after chemical or surgical castration. Furthermore, these data suggest that, perhaps, upstream targets such as the cytokines IL-1β and IL-6 may provide alternative drug targets for inhibiting prostate cancer progression. Prostate 53: 1–8, 2002. © 2002 Wiley-Liss, Inc.

Metastasis is responsible for the vast majority of prostate cancer deaths. As such, it is essential to understand the mechanisms driving prostate cancer to this lethal stage. Deregulation of microRNAs is increasingly implicated in the progression and metastasis of prostate and other cancers. MiR-23b and miR-27b, two members of the same miR cluster (miR-23b/-27b), are down-regulated in human metastatic, castration resistant prostate cancer (CRPC) as compared to primary tumors and benign tissue. However, the role of the miR-23b/-27b cluster is not fully understood, particularly in metastatic disease. Interpretation of existing data is complicated by the analysis of the individual effects of miR-23b and miR-27b even though these miRs are co-transcribed and coordinately expressed. The primary goal of our study is to determine the effects of miR-23b/-27b on metastatic processes in vitro and in vivo. Ectopic expression of miR-23b/-27b in two aggressive prostate cancer cell lines decreases migration, invasion and anchorage-independent growth. Conversely, inhibition of miR-23b/-27b using specific antagomirs in relatively indolent prostate cancer cells promotes increased invasion and migration. E-cadherin protein and mRNA levels were inversely related to miR-23b/-27b levels. In contrast, manipulation of miR-23b/-27b levels had no effect on prostate cancer cell proliferation in any of the tested cell lines. These findings suggest that miR-23b/-27b is specifically linked to metastasis suppression. The Rho GTPase, Rac1, is hyperactive in CRPC cell lines and patient samples. Since Rac1 promotes invasion and migration, we investigated the effects of miR-23b/-27b expression and inhibition on Rac1 activity. Ectopic expression of miR-23b/-27b in aggressive prostate cancer cell lines significantly attenuates active Rac1 while having no effect on total Rac1 levels. Conversely, inhibition of miR-23b/-27b in less aggressive prostate cancer cells increased Rac1 activity but not Rac1 levels. We further examined the effects of miR-23b/-27b on prostate cancer metastasis in vivo. Metastatic prostate cancer cells expressing luciferase and miR-23b/-27b or a scrambled control, were injected into the ventral prostates of nude mice. Orthotopic tumor formation and metastases were assessed by bioluminescence imaging. Metastatic tumor burden was greatly decreased in the tumors derived from miR-23b/-27b expressing cells. Taken together, these data demonstrate that expression of miR-23b/-27b exerts metastasis-suppressing effects in vitro and in vivo. The miR-23b/-27b cluster may be a useful biomarker of poor prognosis in addition to having therapeutic potential in advanced, metastatic prostate cancer. Citation Format: Meghan A. Rice, Reema Ishteiwy, Thirupandiyur Udayakumar, Derek Dykxhoorn, Kerry L. Burnstein. The miR-23b/-27b cluster decreases metastasis of aggressive prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1467. doi:10.1158/1538-7445.AM2014-1467

Background Matrix metalloproteinases (MMPs) are involved in tumor progression. Matrilysin (MMP-7) has been shown to be upregulated in prostatic carcinomas and can increase the invasive capacity of DU-145 cells. Because of the heterogenous nature of prostatic tumors, we examined promatrilysin expression in cocultures containing two different prostatic carcinoma cell lines, DU-145 and LNCaP. Methods Using enzyme linked immunosorbent assay (ELISA) analyses, promatrilysin expression was measured in DU-145/LNCaP cocultures and conditioned media cross-cultures. The effects of blocking IL-6 on promatrilysin expression were examined by pretreating conditioned media with IL-6 neutralizing antibody. Results A significant induction of promatrilysin expression was observed in DU-145/LNCaP cocultures compared to LNCaP cells alone. In addition, DU-145 conditioned medium induced the same fold induction of promatrilysin as was observed in the cocultures. LNCaP cell conditioned medium did not induce promatrilysin expression in DU-145 cells. Neutralization of IL-6 with neutralizing antibody abrogated DU-145 conditioned media induced promatrilysin expression to baseline levels. Conclusions IL-6 secreted by DU-145 cells can induce promatrilysin expression in LNCaP cells. IL-6, in vivo, may act as a paracrine signaling factor that regulates matrix metalloproteinase expression. Therefore, IL-6 may play a role in invasive metastatic processes of a prostate carcinoma. Prostate 48:206–209, 2001. © 2001 Wiley-Liss, Inc.

Bisphosphonates are used for the osteoporosis and in palliative cancer related bone pain management. A role in the cancer cell proliferation and metastases prevention has been proposed. Here, we determined the possibility of utilizing NIR dye conjugated bisphosphonate drug as a potential theragnostic agent. Alendronate was conjugated to water soluble, near-infrared tricarbocyanine, cyclic enamine-functionalized dye, and the imaging probe was used for the purpose. Near-infrared (NIR) fluorescence imaging of both LNCaP cells in cell culture and grown orthotopically in nude mice indicate NIR-bisphosphonate binding. Both in vivo and ex vivo NIRF image analysis of 4 nmol of NIR conjugate delivery show substantial tumor residence time and routine biodistribution to areas of high metabolic bone activity. We present here early evidence of NIR-bisphosphonate analog as a potential diagnostic tool. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5327. doi:10.1158/1538-7445.AM2011-5327