Plasmonic nano-aperture label-free imaging of single small extracellular vesicles for cancer detection.

BACKGROUND: Small extracellular vesicle (sEV) analysis can potentially improve cancer detection and diagnostics. However, this potential has been constrained by insufficient sensitivity, dynamic range, and the need for complex labeling. METHODS: In this study, we demonstrate the combination of PANORAMA and fluorescence imaging for single sEV analysis. The co-acquisition of PANORAMA and fluorescence images enables label-free visualization, enumeration, size determination, and enables detection of cargo microRNAs (miRs). RESULTS: An increased sEV count is observed in human plasma samples from patients with cancer, regardless of cancer type. The cargo miR-21 provides molecular specificity within the same sEV population at the single unit level, which pinpoints the sEVs subset of cancer origin. Using cancer cells-implanted animals, cancer-specific sEVs from 20 µl of plasma can be detected before tumors were palpable. The level plateaus between 5-15 absolute sEV count (ASC) per µl with tumors ≥8 mm3. In healthy human individuals (N = 106), the levels are on average 1.5 ASC/µl (+/- 0.95) without miR-21 expression. However, for stage I-III cancer patients (N = 205), nearly all (204 out of 205) have levels exceeding 3.5 ASC/µl with an average of 12.2 ASC/µl (±9.6), and a variable proportion of miR-21 labeling among different tumor types with 100% cancer specificity. Using a threshold of 3.5 ASC/µl to test a separate sample set in a blinded fashion yields accurate classification of healthy individuals from cancer patients. CONCLUSIONS: Our techniques and findings can impact the understanding of cancer biology and the development of new cancer detection and diagnostic technologies.

Small extracellular vesicles (sEVs) are tiny particles derived from cells that can be detected in bodily fluids such as blood. Detecting sEVs and analyzing their contents may potentially help us to diagnose disease, for example by observing differences in sEV numbers or contents in the blood of patients with cancer versus healthy people. Here, we combine two imaging methods ­ our previously developed method PANORAMA and imaging of fluorescence emitted by sEVs­to visualize and count sEVs, determine their size, and analyze their cargo. We observe differences in sEV numbers and cargo in samples taken from healthy people versus people with cancer and are able to differentiate these two populations based on our analysis of sEVs. With further testing, our approach may be a useful tool for cancer diagnosis and provide insights into the biology of cancer and sEVs.

ANPEP/CD13 Expression as a Marker of Lymphovascular Invasion and Survival in Esophageal Adenocarcinoma.

BACKGROUND: The presence of lymphovascular invasion (LVI) in early esophageal adenocarcinoma (EAC) is associated with more aggressive disease. Molecular markers associated with LVI are still largely unknown. Using a combination of transcriptomic analysis and validation experiments, we sought to describe markers for LVI and survival. METHODS: We performed NanoString expression profiling using RNA from 60 EAC specimens collected from surgery-only cases between 2000 and 2012. Differentially expressed genes (DEGs) were correlated with pathologic characteristics (T and N status and presence of LVI). Kaplan-Meier and Cox regression analyses were used to correlate gene expression with overall survival. Expression of alanyl aminopeptidase, membrane (ANPEP)/CD13 was validated by immunohistochemistry (IHC) in EAC tissue microarray and in EAC cell lines. RESULTS: We identified >20 up-regulated DEGs in tumor samples containing LVI. Multivariable analysis showed depth of invasion and ANPEP/CD13 expression were independently associated with overall survival, whereas nodal status was not. IHC analysis demonstrated overexpression of the ANPEP/CD13 protein in dysplastic Barrett esophagus and EAC tumors. Kaplan-Meier analysis showed that patients with higher RNA expression and strongly positive ANPEP/CD13 membrane IHC-Histoscore staining have shorter survival (P = .002). Down-regulation of ANPEP/CD13 expression by short hairpin RNA vector reduces colony formation, migration, and invasion of FLO-1 EAC cells. Overexpression of CD13 in SKGT4 EAC cells increases colony formation, motility, and invasion in vitro. CONCLUSIONS: Elevated expression of ANPEP/CD13 indicates shorter survival of EAC patients and a more invasive phenotype of cancer cells in vitro. Validation in a larger sample group is required to better understand the clinical significance of ANPEP/CD13 and other candidate genes.

MMTV promoter-regulated caveolin-1 overexpression yields defective parenchymal epithelia in multiple exocrine organs of transgenic mice.

Caveolin-1 (Cav-1) is a major structural protein of caveolae, specialized plasma membrane invaginations that are involved in a cell-specific fashion in diverse cell activities such as molecular transport, cell adhesion, and signal transduction. In normal adult mammals, Cav-1 expression is abundant in mesenchyme-derived cells but relatively low in epithelial parenchyma. However, epithelial Cav-1 overexpression is associated with development and/or progression of many carcinomas. In this study, we generated and characterized a transgenic mouse model of Cav-1 overexpression under the control of a mouse mammary tumor virus (MMTV) long terminal-repeat promoter, which is predominantly expressed in specific epithelial cells. The MMTVcav-1(+) transgenic mice were fertile, and females bore litters of normal size with no obvious developmental abnormalities. However, by age 11months, the MMTVcav-1(+) mice demonstrated overtly different phenotypes in multiple exocrine organs when compared with their nontransgenic MMTVcav-1(-) littermates. Cav-1 overexpression in MMTVcav-1(+) mice produced organ-specific abnormalities, including hypotrophy of mammary glandular epithelia, bronchiolar epithelial hyperplasia and atypia, mucous-cell hyperplasia in salivary glands, elongated hair follicles and dermal thickening in the skin, and reduced accumulation of enzymogen granules in pancreatic acinar cells. In addition, the MMTVcav-1(+) transgenic mice tended to have a greater incidence of malignant tumors, including lung and liver carcinomas and lymphoma, than their MMTVcav-1(-) littermates. Our results indicate that Cav-1 overexpression causes organ-specific, age-related epithelial disorders and suggest the potential for increased susceptibility to carcinogenesis.

HER2 Confers Resistance to Foretinib Inhibition of MET-Amplified Esophageal Adenocarcinoma Cells.

BACKGROUND: Recent genomic studies indicated that esophageal adenocarcinoma (EAC) is driven by amplification of c-MET or HER2 or both in a subset of patients. We studied the effect of MET targeting by the small molecule inhibitor foretinib in EAC cells and the interplay between MET and HER2 signaling. METHODS: We measured the expression levels and phosphorylation status of MET and HER2 proteins in EAC cell lines using Western blot analysis. The expression levels of MET and HER2 were manipulated by transfecting cells with specific siRNA or a plasmid expressing HER2. The small molecule inhibitors of c-MET and ERBB1/2 (foretinib and lapatinib, respectively) were tested for effect on growth, apoptosis, and downstream signaling pathways of EAC cells as single agents or in combination. The response to inhibitors was correlated to the levels of MET, HER2 expression, and amplification status. RESULTS: Foretinib inhibits phosphorylation of MET, which correlated with reduced EAC cell growth and inhibition of AKT and ERK phosphorylation. Cell growth inhibition by foretinib is most profound in the ESO51 cell line, which has MET gene amplification and overexpression. Inhibition of MET signaling by foretinib or siRNA-specific knock down of MET expression induces apoptosis in ESO51 cells. Ectopic expression of HER2 reduces foretinib-mediated growth inhibition and downstream ERK phosphorylation in ESO51-HER2 cells. The EAC OE33 cell line, with amplification and overexpression of both MET and HER2, demonstrated reduced sensitivity to foretinib or lapatinib and had a transient effect on downstream inhibition of phosphorylated AKT and ERK (p-AKT, p-ERK). The coadministration of foretinib and lapatinib effectively blocked both MET and HER2 signaling through the p-AKT and p-ERK pathways, dramatically inhibited growth, and induced apoptosis to overcome single-agent resistance in OE33 cells. CONCLUSIONS: The mechanism for foretinib growth inhibition in MET-amplified EAC tumor cells is demonstrated. The interplay of dual MET/HER2 overexpression in the AKT and ERK pathways for esophageal cancer is described. Therefore, combination therapy could be a novel strategy for EAC with amplification of both MET and HER2.

Outcomes after endoscopic mucosal resection or esophagectomy for submucosal esophageal adenocarcinoma.

OBJECTIVES: Endoscopic mucosal resection (EMR) is a diagnostic and potentially therapeutic option for patients with submucosal esophageal adenocarcinoma. However, there are significant concerns regarding the risk of lymph node metastasis. Our purpose was to construct a comparative effectiveness analysis comparing recurrence patterns after therapeutic EMR or esophagectomy. METHODS: Patients who underwent therapeutic EMR or esophagectomy from 2007 to 2015 with pathologically staged submucosal adenocarcinoma were identified from a departmental database. Cancer-related outcomes were compared among an unmatched as well as a propensity matched cohort. Risk stratification was also used to compare results among those with a low, medium, or high risk of nodal metastasis. RESULTS: Seventy-two patients met criteria for analysis, among whom 23 underwent therapeutic EMR with esophageal preservation and 49 underwent esophagectomy. Median follow-up was 43 months. Patients who underwent esophagectomy had larger, deeper tumors. Esophageal preservation was associated with an increased risk of local recurrence (P = .01), but not distant recurrence (P = .44). After propensity matching, there continued to be no difference in distant recurrence rate (P = .66). In a risk-stratified analysis, low-risk patients showed no recurrences or cancer-related deaths, however, high-risk patients showed a trend toward increased distant recurrence after therapeutic EMR. CONCLUSIONS: Esophageal preservation after therapeutic EMR was associated with an increased risk of local recurrence. Among low-risk patients, either strategy resulted in excellent cancer control. However, among high-risk patients, esophageal preservation showed a trend toward increased distant failure. These findings should prompt further investigation to determine optimal treatment for patients with submucosal esophageal adenocarcinoma.

mRTVP-1, a novel p53 target gene with proapoptotic activities.

We identified a novel mouse gene, mRTVP-1, as a p53 target gene using differential display PCR and extensive promoter analysis. The mRTVP-1 protein has 255 amino acids and differs from the human RTVP-1 (hRTVP-1) protein by two short in-frame deletions of two and nine amino acids. RTVP-1 mRNA was induced in multiple cancer cell lines by adenovirus-mediated delivery of p53 and by gamma irradiation or doxorubicin both in the presence and in the absence of endogenous p53. Analysis of RTVP-1 expression in nontransformed and transformed cells further supported p53-independent gene regulation. Using luciferase reporter and electrophoretic mobility shift assays we identified a p53 binding site within intron 1 of the mRTVP-1 gene. Overexpression of mRTVP-1 or hRTVP-1 induced apoptosis in multiple cancer cell lines including prostate cancer cell lines 148-1PA, 178-2BMA, PC-3, TSU-Pr1, and LNCaP, a human lung cancer cell line, H1299, and two isogenic human colon cancer cell lines, HCT116 p53(+/+) and HCT116 p53(-/-), as demonstrated by annexin V positivity, phase-contrast microscopy, and in selected cases 4',6'-diamidino-2-phenylindole staining and DNA fragmentation. Deletion of the signal peptide from the N terminus of RTVP-1 reduced its apoptotic activities, suggesting that a secreted and soluble form of RTVP-1 may mediate, in part, its proapoptotic activities.

RTVP-1, a tumor suppressor inactivated by methylation in prostate cancer.

We previously identified and characterized a novel p53-regulated gene in mouse prostate cancer cells that was homologous to a human gene that had been identified in brain cancers and termed RTVP-1 or GLIPR. In this report, we document that the human RTVP-1 gene is also regulated by p53 and induces apoptosis in human prostate cancer cell lines. We show that the expression of the human RTVP-1 gene is down-regulated in human prostate cancer specimens compared with normal human prostate tissue at the mRNA and protein levels. We further document epigenetic changes consistent with RTVP-1 being a tumor suppressor in human prostate cancer.

TGF-ß/ß2-spectrin/CTCF-regulated tumor suppression in human stem cell disorder Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is a human stem cell disorder, and individuals with this disease have a substantially increased risk (~800-fold) of developing tumors. Epigenetic silencing of ß2-spectrin (ß2SP, encoded by SPTBN1), a SMAD adaptor for TGF-ß signaling, is causally associated with BWS; however, a role of TGF-ß deficiency in BWS-associated neoplastic transformation is unexplored. Here, we have reported that double-heterozygous Sptbn1+/- Smad3+/- mice, which have defective TGF-ß signaling, develop multiple tumors that are phenotypically similar to those of BWS patients. Moreover, tumorigenesis-associated genes IGF2 and telomerase reverse transcriptase (TERT) were overexpressed in fibroblasts from BWS patients and TGF-ß-defective mice. We further determined that chromatin insulator CCCTC-binding factor (CTCF) is TGF-ß inducible and facilitates TGF-ß-mediated repression of TERT transcription via interactions with ß2SP and SMAD3. This regulation was abrogated in TGF-ß-defective mice and BWS, resulting in TERT overexpression. Imprinting of the IGF2/H19 locus and the CDKN1C/KCNQ1 locus on chromosome 11p15.5 is mediated by CTCF, and this regulation is lost in BWS, leading to aberrant overexpression of growth-promoting genes. Therefore, we propose that loss of CTCF-dependent imprinting of tumor-promoting genes, such as IGF2 and TERT, results from a defective TGF-ß pathway and is responsible at least in part for BWS-associated tumorigenesis as well as sporadic human cancers that are frequently associated with SPTBN1 and SMAD3 mutations.

Combined therapeutic effects of adenoviral vector-mediated GLIPR1 gene therapy and radiotherapy in prostate and bladder cancer models.

OBJECTIVES: The objectives of this study are to explore the potential benefits of combining AdGlipr1 (or AdGLIPR1) gene therapy with radiotherapy using subcutaneous prostate and bladder cancer models. MATERIALS AND METHODS: Combination adenoviral vector-mediated gene therapy and radiotherapy were applied to 178-2 BMA and TSU-Pr1 cells in vitro and colony formation and apoptosis were analyzed. In addition, combination therapies were administered to mice bearing subcutaneous 178-2 BMA and TSU-Pr1 tumors, and tumor growth suppression and survival extension were compared with the monotherapies (AdGlipr1/AdGLIPR1 and radiotherapy) or control vector Adv/CMV/ßgal, as well as single-cycle treatment with 2-cycle treatment. RESULTS: Combination treatment significantly suppressed colony formation and increased apoptosis in vitro. In vivo, combination therapy produced significant 178-2 BMA and TSU-Pr1 tumor growth suppression and survival extension compared with the monotherapies or the control. Further tumor growth suppression and survival extension were observed after 2 cycles of the combination treatment. CONCLUSIONS: Combining AdGlipr1 (AdGLIPR1) with radiotherapy may achieve additive or synergistic tumor control in selected prostate and bladder tumors, and additional therapeutic effects may result with repeated treatment cycles.

Serum caveolin-1, a biomarker of drug response and therapeutic target in prostate cancer models.

We investigated the effect of dasatinib and sunitinib on tyrosine kinase (TK) signaling, caveolin-1 (Cav-1) expression and secretion and proliferation of PC-3 and DU145 prostate cancer cells in vitro and in vivo. Treatment of both cell lines with either dasatinib or sunitinib reduced phosphorylation of PDGFR, VEGFR2, Akt, FAK, Src (dasatinib only) and Cav-1, and reduced cellular and secreted levels of Cav-1. Both agents dose-dependently inhibited proliferation of these cells. In PC-3 and DU145 subcutaneous xenografts, treatment with dasatinib, sunitinib or anti-Cav-1 antibody (Ab) alone produced significant tumor regression compared with that by vehicle or IgG alone. Combined dasatinib and anti-Cav-1 Ab treatment or sunitinib and anti-Cav-1 Ab produced greater tumor regression than either treatment alone. Serum Cav-1 levels were lower in dasatinib- and sunitinib-treated mice than they were in vehicle-treated mice, and correlated positively with tumor growth in dasatinib- and sunitinib-treated groups (r = 0.48, p = 0.031; r = 0.554, p = 0.0065, respectively), compared with vehicle controls. Cav-1 knockdown, in combination with dasatinib or sunitinib treatment in PC-3 cells, caused a greater reduction in the phosphorylation of PDGFR-ß and VEGFR2, and expression and secretion of PDGF-B and VEGF-A than that in PC-3 cells treated with dasatinib or sunitinib alone in control siRNA cells, suggesting that Cav-1 is involved in an autocrine pathway that is affected by these drugs. Overall, our results suggest a role for Cav-1 as a biomarker of response to both dasatinib and sunitinib treatment and as a therapeutic target in prostate cancer.

Caveolin-1-LRP6 signaling module stimulates aerobic glycolysis in prostate cancer.

Caveolin 1 (Cav-1) is a plasma membrane-associated protein with the capacity to modulate signaling activities in a context-dependent fashion. Interactions between Cav-1 and low-density lipoprotein receptor-related protein 6 (LRP6) were reported to be important for the regulation of Wnt-ß-catenin (ß-cat) signaling. Cav-1 also interacts with insulin and IGF-I receptors (IGF-IR/IR) and can stimulate IR kinase activities. We found positive correlation between Cav-1 and LRP6 expression in both human primary prostate cancer and metastasis tissues and in PC-3 cells. Cav-1 stimulation of Wnt-ß-cat signaling and c-Myc levels was positively associated with LRP6 expression in LNCaP, PC-3, and DU145 prostate cancer cells. Importantly, LRP6 and, to a lesser extent, Cav-1 were found to stimulate aerobic glycolysis. These activities were positively associated with the expression of HK2 and Glut3 and shown to be dependent on Akt signaling by both gene knockdown and chemical inhibition methods. We further showed that Cav-1 and LRP6 exert their effects on Akt and glycolytic activities by stimulating IGF-IR/IR signaling. Overall, our results show that Cav-1 interacts with LRP6 to generate an integrated signaling module that leads to the activation of IGF-IR/IR and results in stimulation of Akt-mTORC1 signaling and aerobic glycolysis in prostate cancer.

Glioma pathogenesis-related protein 1 induces prostate cancer cell death through Hsc70-mediated suppression of AURKA and TPX2.

In this study we report that expression of glioma pathogenesis-related protein 1 (GLIPR1) regulated numerous apoptotic, cell cycle, and spindle/centrosome assembly-related genes, including AURKA and TPX2, and induced apoptosis and/or mitotic catastrophe (MC) in prostate cancer (PCa) cells, including p53-mutated/deleted, androgen-insensitive metastatic PCa cells. Mechanistically, GLIPR1 interacts with heat shock cognate protein 70 (Hsc70); this interaction is associated with SP1 and c-Myb destabilization and suppression of SP1- and c-Myb-mediated AURKA and TPX2 transcription. Inhibition of AURKA and TPX2 using siRNA mimicked enforced GLIPR1 expression in the induction of apoptosis and MC. Recombinant GLIPR1-ΔTM protein inhibited AURKA and TPX2 expression, induced apoptosis and MC, and suppressed orthotopic xenograft tumor growth. Our results define a novel GLIPR1-regulated signaling pathway that controls apoptosis and/or mitotic catastrophe in PCa cells and establishes the potential of this pathway for targeted therapies.

Caveolin-1 upregulation contributes to c-Myc-induced high-grade prostatic intraepithelial neoplasia and prostate cancer.

Previously we reported caveolin-1 (Cav-1) overexpression in prostate cancer cells and showed that it promotes prostate cancer progression. Here, we report that Cav-1 was overexpressed in 41.7% (15 of 36) of human high-grade prostatic intraepithelial neoplasia (HGPIN) specimens obtained during radical prostatectomies. Positive correlations exist between Cav-1-positive (Cav-1(+)) HGPIN and Cav-1(+) primary prostate cancer (rho = 0.655, P < 0.0001) and between Cav-1 and c-Myc expression in HGPIN (rho = 0.41, P = 0.032). To determine whether Cav-1 cooperates with c-Myc in development of premalignant lesions and prostate cancer in vivo, we generated transgenic mice with c-Myc overexpression driven by the ARR(2)PB promoter. In this ARR(2)PB-c-myc model, Cav-1 overexpression was found in mouse PIN (mPIN) lesions and prostate cancer cells and was associated with a significantly higher ratio of proliferative to apoptotic labeling in mPIN lesions than in the Cav-1-negative epithelia adjacent to those lesions (10.02 vs. 4.34; P = 0.007). Cav-1 overexpression was also associated with increased levels of P-Akt and VEGF-A, which were previously associated with Cav-1-induced prostate cancer cell survival and positive feedback regulation of cellular Cav-1 levels, respectively. In multiple prostate cancer cell lines, Cav-1 protein (but not mRNA) was induced by c-Myc transfection, whereas VEGF siRNA transfection abrogated c-Myc-induced Cav-1 overexpression, suggesting a c-Myc-VEGF-Cav-1 signaling axis. Overall, our results suggest that Cav-1 is associated with c-Myc in the development of HGPIN and prostate cancer. Furthermore, Cav-1 overexpression in HGPIN is potentially a biomarker for early identification of patients who tend to develop Cav-1(+) primary prostate cancer.

Castration-induced changes in mouse epididymal white adipose tissue.

We analyzed the effects of castration on epididymal white adipose tissue (WAT) in C57BL/6J mice which were fed a regular or high-fat diet. Fourteen days following surgical castration profound effects on WAT tissue such as reductions in WAT wet weight and WAT/body weight ratio, induction of lipolysis and morphologic changes characterized by smaller adipocytes, and increased stromal cell compartment were documented in both dietary groups. Castrated animals had decreased serum leptin levels independent of diet but diet-dependent decreases in serum adiponectin and resistin. The castrated high-fat group had dramatically lower serum triglyceride levels. Immunohistochemical analysis revealed higher staining for smooth muscle actin, macrophage marker Mac-3, and Cxcl5 in the castrated than in the control mice in both dietary groups. We also detected increased fatty-acid synthase expression in the stromal compartment of WAT in the regular-diet group. Castration also reduces the expression of androgen receptor in WAT in the regular-diet group. We conclude that castration reduces tissue mass and affects biologic function of WAT in mice.

GLIPR1 suppresses prostate cancer development through targeted oncoprotein destruction.

Downregulation of the proapoptotic p53 target gene glioma pathogenesis-related protein 1 (GLIPR1) occurs frequently in prostate cancer, but the functional meaning of this event is obscure. Here, we report the discovery of functional relationship between GLIPR1 and c-Myc in prostate cancer where c-Myc is often upregulated. We found that the expression of GLIPR1 and c-Myc were inversely correlated in human prostate cancer. Restoration of GLIPR1 expression in prostate cancer cells downregulated c-myc levels, inhibiting cell-cycle progression. Downregulation was linked to a reduction in ß-catenin/TCF4-mediated transcription of the c-myc gene, which was caused by GLIPR1-mediated redistribution of casein kinase 1α (CK1α) from the Golgi apparatus to the cytoplasm where CK1α could phosphorylate ß-catenin and mediate its destruction. In parallel, GLIPR1 also promoted c-Myc protein ubiquitination and degradation by glycogen synthase kinase-3α- and/or CK1α-mediated c-Myc phosphorylation. Notably, genetic ablation of the mouse homolog of Glipr1 cooperated with c-myc overexpression to induce prostatic intraepithelial neoplasia and prostate cancer. Together, our findings provide evidence for CK1α-mediated destruction of c-Myc and identify c-Myc S252 as a crucial CK1α phosphorylation site for c-Myc degradation. Furthermore, they reveal parallel mechanisms of c-myc downregulation by GLIPR1 that when ablated in the prostate are sufficient to drive c-Myc expression and malignant development.

GLIPR1 tumor suppressor gene expressed by adenoviral vector as neoadjuvant intraprostatic injection for localized intermediate or high-risk prostate cancer preceding radical prostatectomy.

BACKGROUND: GLIPR1 is upregulated by p53 in prostate cancer cells and has preclinical antitumor activity. A phase I clinical trial was conducted to evaluate the safety and activity of the neoadjuvant intraprostatic injection of GLIPR1 expressing adenovirus for intermediate or high-risk localized prostate cancer before radical prostatectomy (RP). METHODS: Eligible men had localized prostate cancer (T1-T2c) with Gleason score greater than or equal to 7 or prostate-specific antigen 10 ng/mL or more and were candidates for RP. Patients received the adenoviral vector expressing the GLIPR1 gene by a single injection into the prostate followed four weeks later by RP. Six viral particle (vp) dose levels were evaluated: 10(10), 5 × 10(10), 10(11), 5 × 10(11), 10(12), and 5 × 10(12) vp. RESULTS: Nineteen patients with a median age of 64 years were recruited. Nine men had T1c, 4 had T2a, and 3 had T2b and T2c clinical stage. Toxicities included urinary tract infection (n = 3), flu-like syndrome (n = 3), fever (n = 1), dysuria (n = 1), and photophobia (n = 1). Laboratory toxicities were grade 1 elevated AST/ALT (n = 1) and elevations of PTT (n = 3, with 1 proven to be lupus anticoagulant). No pathologic complete remission was seen. Morphologic cytotoxic activity, induction of apoptosis, and nuclear p27(Kip1) upregulation were observed. Peripheral blood CD8(+), CD4(+), and CD3(+) T-lymphocytes were increased, with upregulation of their HLA-DR expression and elevations of serum IL-12. CONCLUSIONS: The intraprostatic administration of GLIPR1 tumor suppressor gene expressed by an adenoviral vector was safe in men, with localized intermediate or high-risk prostate cancer preceding RP. Preliminary evidence of biologic antitumor activity and systemic immune response was documented.

Caveolin-1 promotes autoregulatory, Akt-mediated induction of cancer-promoting growth factors in prostate cancer cells.

Caveolin-1 (cav-1) and the cancer-promoting growth factors vascular endothelial growth factor (VEGF), transforming growth factor beta1 (TGF-beta1), and fibroblast growth factor 2 (FGF2) are often found to be upregulated in advanced prostate cancer and other malignancies. However, the relationship between cav-1 overexpression and growth factor upregulation remains unclear. This report presents, to our knowledge, the first evidence that in prostate cancer cells, a positive autoregulatory feedback loop is established in which VEGF, TGF-beta1, and FGF2 upregulate cav-1, and cav-1 expression, in turn, leads to increased levels of VEGF, TGF-beta1, and FGF2 mRNA and protein, resulting in enhanced invasive activities of prostate cancer cells, i.e., migration and motility. Our results further show that cav-1-enhanced mRNA stability is a major mechanism underlying the upregulation of these cancer-promoting growth factors, and that PI3-K-Akt signaling is required for forming this positive autoregulatory feedback loop.

Functional analysis of secreted caveolin-1 in mouse models of prostate cancer progression.

Previously, we reported that caveolin-1 (cav-1) is overexpressed in metastatic prostate cancer and that virulent prostate cancer cells secrete biologically active cav-1. We also showed that cav-1 expression leads to prosurvival activities through maintenance of activated Akt and that cav-1 is taken up by other cav-1-negative tumor cells and/or endothelial cells, leading to stimulation of angiogenic activities through PI-3-K-Akt-eNOS signaling. To analyze the functional consequences of cav-1 overexpression on the development and progression of prostate cancer in vivo, we generated PBcav-1 transgenic mice. Adult male PBcav-1 mice showed significantly increased prostatic wet weight and higher incidence of epithelial hyperplasia compared with nontransgenic littermates. Increased immunostaining for cav-1, proliferative cell nuclear antigen, P-Akt, and reduced nuclear p27(Kip1) staining occurred in PBcav-1 hyperplastic prostatic lesions. PBcav-1 mice showed increased resistance to castration-induced prostatic regression and elevated serum cav-1 levels compared with nontransgenic littermates. Intraprostatic injection of androgen-sensitive, cav-1-secreting RM-9 mouse prostate cancer cells resulted in tumors that were larger in PBcav-1 mice than in nontransgenic littermates (P = 0.04). Tail vein inoculation of RM-9 cells produced significantly more experimental lung metastases in PBcav-1 males than in nontransgenic male littermates (P = 0.001), and in cav-1(+/+) mice than in cav-1(-/-) mice (P = 0.041). Combination treatment with surgical castration and systemic cav-1 antibody dramatically reduced the number of experimental metastases. These experimental data suggest a causal association of secreted cav-1 and prostate cancer growth and progression.

Tumor cell-secreted caveolin-1 has proangiogenic activities in prostate cancer.

Caveolin, a major structural component of specialized plasma membrane invaginations (caveolae) that participate in diverse cellular activities, has been implicated in the pathogenesis of several human diseases, including cancer. We showed in earlier studies that caveolin-1 (cav-1) is consistently and strongly overexpressed in metastatic prostate cancer and is secreted in a biologically active form by virulent prostate cancer cells. Using both in vitro and in vivo model systems, we now present evidence supporting a proangiogenic role for cav-1 in prostate cancer development and progression. Recombinant cav-1 (rcav-1) was taken up by cav-1(-/-) endothelial cells through either a lipid raft/caveolae- or clathrin-dependent mechanism, leading to specific angiogenic activities (tubule formation, cell migration, and nitric oxide production) that were mediated by rcav-1 stimulation of the PI3K-Akt-eNOS signaling module. Pathologic angiogenesis induced by cav-1 in prostate cancer-bearing mice correlated with an increased frequency, number, and size of lung metastases. We propose that in addition to its antiapoptotic role, cav-1 secreted by prostate cancer cells functions critically as a proangiogenic factor in metastatic progression of this tumor. These new insights into cav-1 function in prostate cancer may provide a base for the design of clinically applicable therapeutic strategies.