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Diaphanous-related formin-3 (DIAPH3), a cytoskeletal regulator of the formin family, is lost at high frequency in metastatic breast and prostate cancers and has characteristics of a non-canonical metastasis suppressor. We previously showed that DIAPH3 silencing evokes a transition to an “amoeboid” phenotype, characterized by rapid motility, RhoA/ROCK and ERK pathway activation, dynamic membrane blebbing and increased shedding of microvesicles. Here we demonstrate that DIAPH3 loss reduces microtubule (MT) stability in prostate and breast cancer cell lines, and increases the cells' sensitivity to the taxanes, paclitaxel and docetaxel and the non-taxane, epothilone B. DIAPH3 silencing reduces membrane localization of the multidrug resistance-associated protein, MRP1, a regulator of taxane efflux, and increases intracellular accumulation of fluorescent paclitaxel. Baseline acetylation of tubulin, which is proportional to the number of stable MT, is lower in DIAPH3-silenced cells. In contrast, the fold-increase of tubulin acetylation after treatment with MT stabilizing drugs is greater in DIAPH3 knockdown compared to control cells. Further, cytotoxicity induced by paclitaxel, docetaxel or epothilone B is enhanced by DIAPH3 silencing. Analysis of gene expression profiles of breast cancer patients treated with chemotherapeutic regimens containing taxanes in a neoadjuvant phase-2 clinical trial revealed that low DIAPH3 expression is associated with increased relapse free survival. Collectively, the data demonstrate that DIAPH3 deficiency increases responsiveness to widely used MT stabilizing drugs. These findings have implications for the inclusion of DIAPH3 in biomarker panels that stratify patients for treatment with taxanes, in particular in the neoadjuvant setting. Citation Format: Samantha Morley, Sara G. Pollan, Sungyong You, Beatrice S. Knudsen, Michael Freeman. The formin, DIAPH3, regulates response to MT stabilizing drugs in prostate and breast 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 5474. doi:10.1158/1538-7445.AM2014-5474

Laminin binding integrins, A3B1 and A6B1 drive in part, cancer cell migration and metastasis. Cell migration requires dynamic internalization and recycling of surface integrins. However, whether laminin binding integrin internalization is coordinately or independently controlled is not known. In this study, we quantitated the internalization of integrin A3 and A6 and whether crosstalk influences their respective internalization and subsequent cancer cell migration. We quantitated the internalization of A6 and A3 integrins, using fluorophore conjugated antibody based internalization assays and flow cytometry. In the prostate cancer cell line DU145, we observed constitutive internalization of integrin A6 with a surface half-life of approximately 30 minutes as compared to >70 minutes for integrin A3, 45 minutes for unrelated integrin Av or 10 minutes for the unrelated transferrin receptor. Silencing A3 integrin expression resulted in a 10 percent increase in the total amount of internalized A6 integrin, with a decreased surface half-life of 20 minutes. The internalized A6 integrin showed intracellular punctate staining colocalized with the early endosome marker EEA1 and Rab4 containing vesicles. Increased integrin A6 internalization led to 40% increase in migration which was dependent on A6B1 integrin. Silencing integrin A6 expression, however, did not affect the internalization of A3 integrin indicating a unidirectional regulation of integrin internalization. The changes in the integrin internalization could be clearly ascribed to alpha subunits, as the obtained half-life of A6 and A3 integrins were independent of antibody used or integrin engagement by ligand mimetic peptides. Taken together, these data indicate that A6B1 and A3B1 integrins have different internalization kinetics and the presence of integrin A3 decreased integrin A6 internalization and ensuing cell migration. These data are consistent with previous observations in normal systems suggesting that A3B1 provides a provisional matrix during early stages of wound healing, requiring subsequent stable adhesion complexes and integrin A6 function. In cancer, the loss of A3 integrin and the increased internalization of A6 integrin may account for the “wounds that won't heal” phenotype and provide a strategy for biological intervention to block tumor progression. (Supported in part by NIH Grants CA23074 and CA159406 and the TACMASS core service of the Arizona Cancer Center) Citation Format: Lipsa Das, Todd A. Anderson, Jaime M.c. Gard, Isis C. Sroka, Stephanie R. Strautman, Raymond B. Nagle, Beatrice S. Knudsen, Anne Cress. A6B1 integrin internalization is increased by loss of A3B1 expression and promotes migration of human prostate cancer cells. [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 4979. doi:10.1158/1538-7445.AM2014-4979

Background: Alcoholic chronic pancreatitis (ACP) is a major risk factor for pancreatic cancer; however, the mechanism through which it promotes cancer is not known. In this study we developed a mouse model of pancreatic cancer induced by ACP and analyzed the effect of the interaction between macrophages and pancreatic cancer cells on progression of the disease. Methods: Pdx1-Cre;LSL-Kras transgenic and wild-type (WT) mice were fed Lieber DeCarli control or alcohol diet for 6 weeks. Mice were subjected to 7-hourly cerulein injections (50µg/kg) twice a week for the last 3 weeks of the diet to induce chronic pancreatitis. Pancreatic intraepithelial neoplastic (PanIN) lesions, proliferation, fibrosis, inflammation, EMT and stem cell markers were analyzed by immunohistochemistry and Western. Monocytes/ macrophages from mice and their conditioned medium were applied to the pancreatic cancer cell line MIA PaCa-2. Cell survival and apoptosis were measured by MTT and ELISA. Expression of cytokines/ chemokines and growth factors by cancer cells and macrophages was measured by multiplex assay analysis. Results: ACP increased fibrosis, stellate cell activation and inflammation in both WT and transgenic mouse pancreata. The increases were significantly greater in the transgenic mice. Further, ACP increased the number and stage of PanIN lesions in the pancreata of transgenic mice. EMT markers were stimulated by ACP in both WT and transgenic mice. Markers of type 2 macrophages (M2) were greater in ACP model compared to control mice. M2 macrophages conditioned media stimulated proliferation, EMT and cancer stemness in cancer cells. Cancer cells or PanIN cells conditioned media stimulated transformation of macrophages to the type 2 phenotype. Conclusion: The results indicate that ACP promotes pancreatic cancer progression in transgenic mice. Our data suggest ACP as well as PanIN and cancer cells stimulate the phenotype change of macrophages toward type 2, which then stimulates cancer cell proliferation, EMT and cancer stemness. These results suggest that tissue macrophages play a key role in the increased risk for pancreatic cancer in patients with alcoholic pancreatitis. Citation Format: Mouad Edderkaoui, Paul Grippo, Hajar Benhaddou, Yassine Ouhaddi, Hidekazu Tsukamoto, Steven Swartwood, Beatrice Knudsen, Helen S. Goodridge, Aida Habtezion, Vay-Liang Go, Stephen J. Pandol. Alcoholic chronic pancreatitis increases tissue transformed macrophages (M2) which promote pancreatic cancer lesions in a Kras mouse model. [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 4847. doi:10.1158/1538-7445.AM2014-4847

Aim: Cabozantinib (XL184) is a potent multi-targeted tyrosine kinase inhibitor (TKI) that is under active investigation in several malignancies including prostate cancer (PC). The mechanism of action in PC has not been well characterized at this time. While considerable attention has been given to the impact of this agent on Tc-bone scans, less is known about its activity in men with visceral metastases. Methods: This study was designed as a single arm phase 2 study of cabozantinib 60 mg daily for men with visceral disease. The primary endpoint of this study is clinical benefit rate at 12 weeks (CBR12) which included stable disease and radiographic response. PSA was not used as an endpoint. Beyond standard clinical assessments, blood and urine were collected for circulating tumor cell (CTC) assessments using CellSearch (CS) and NanoVelcro (NV) chips and large oncosome selection. Biopsies before treatment and at progression are being collected when possible. Circulating tumor cell RNA and large oncosome content were profiled at baseline and 2 weeks for all patients. Results: Eight pts. have been treated of the anticipated 40 patient cohort. 13% had pulmonary metastatic lesion; 88% had hepatic metastases; 88% had concurrent osseous metastases. CBR12 was 83% at the time of this report. Two patients discontinued treatment without evidence of progression due to grade 3 toxicity (diarrhea, corneal defect). Discordant responses were noted between visceral metastases and bone scans. Other grade 3 toxicities included hypertension, hand-foot syndrome, and GGT. CTC numbers by NV and CS declined within 2 weeks and increased at progression. Updated results of CTC and oncosome analysis will be presented including alterations in MET and SChLAP1 expression. Conclusions: Cabozantinib is a TKI with activity in PC even in men with visceral metastasis who are typically considered to have more aggressive disease. The activity in the viscera appears to be lower than that seen in osseous metastases. Further understanding of the biology of cabozantinib based on CTC, oncosomes, and tissue studies will refine our use of this active agent. Disclosure: L.W.K. Chung: research funding from Exelixis. All other authors have declared no conflicts of interest.

e16080 Background: Cabozantinib (XL184) is a potent multi-targeted tyrosine kinase inhibitor (TKI) that is under active investigation in several malignancies including prostate cancer (PC). The mec...

Background: Histone deacetylases (HDAC) inhibitors have been shown to suppress tumor growth in many cancers. HDAC class 1 regulates pancreatic cancer growth and resistance to cell death through a mechanism that involves NF-kB. We have developed a mouse model of pancreatic cancer induced by cigarette smoke. We hypothesized that HDAC-1 mediates progression of the disease induced by smoking and that inhibition of HDAC-1 will prevent both cancer cell survival and EMT/metastasis in vitro and in vivo. Methods: Pdx-Cre;LSLKras mice were exposed to filtered air or to cigarette smoke in smoke chambers for 6 weeks. A set of mice were injected with HDAC1 inhibitor SAHA (50mg/Kg) 5 times per week. Pancreatic intraepithelial neoplasia (PanIN) lesions, fibrosis, and inflammation weremeasured by immunohistochemistry (IHC). Pancreas levels of proliferation, apoptosis, and EMT markers were measured by IHC andWestern. Pharmacological and molecular inhibitors of HDAC1 were applied in MIA PaCa-2 and AsPC-1 cell lines. Inhibition of Zeb1, a regulator of EMT, was achieved by using Zeb1 siRNA. EMT markers and transcription factors as well as proteins acetylation level were measured by Western. Cell survival and apoptosis in cells were measured by MTT assay and DNA fragmentation ELISA assay, respectively. Results: Mice exposed to cigarette smoke showed increased level of PanIN lesions, fibrosis, and inflammation. The three pro-cancer effects were prevented in mice exposed to HDAC-1 inhibitor SAHA. SAHA decreased the levels of PanIN lesions and fibrosis, not only to a level lower than in mice exposed to cigarette smoke, but also lower than in control mice exposed to filtered air. SAHA decreased the protein level of vimentin, zeb1, sox2 and CD133 and increased E-cadherin level in pancreatic tissue indicting that it prevented EMT and stemness. Pharmacological and molecular inhibitions of HDAC-1 also significantly and dose-dependently decreased proliferation and at a lesser extent stimulated apoptosis in pancreatic cancer cells. Zeb1 siRNA decreased the level of vimentin and increased E-cadherin in pancreatic cancer cells. Conclusion: We found that smoking stimulates an increase in PanIN lesions through a mechanism that involves HDAC-1 in Kras transgenic mice. HDAC-1 not only affected proliferation of the cancer cells, but also EMT, which is involved in metastasis and cancer stemness. These data suggest an important role for HDAC-1 in pancreatic carcinogenesis.

93 Background: A key eligibility criterion in many active surveillance (AS) programs is that the biopsy exhibit only Gleason pattern 3 (G3) for a Gleason score of 6 or less. However, 25 to 35% of biopsy Gleason 6 is upgraded to Gleason 7 or higher in the prostatectomy (RP) specimen. Thus, there is a great need for biomarkers that, when measured on G3 tissue in a Gleason 6 biopsy, can predict the presence of unsampled higher grade tumor in the whole prostate. We evaluated PTEN loss by immunohistochemistry (IHC), and PTEN deletion, chromosome 8q (MYC) gain and 8p (LPL) loss by fluorescence in situ hybridization (FISH) for their ability to predict unsampled G4 tumor. Methods: A tissue microarray (TMA) was constructed of RP tissue from three groups of patients (n=50 per group) whose prostates exhibited only Gleason 3+3, only 3+4, or only 4+3 tumor, matched on age, year of RP, and race. In each patient, multiple cores sampled only from areas of G3 were evaluated for PTEN deletion by FISH, PTEN loss by IHC, and chromosome 8p/8q alterations by FISH. Biomarker results were compared between Gleason 6 versus 7 tumors using conditional logistic regression. Results: Patients underwent RP in 2001 to 2009, had median age 60, and median prostate-specific antigen 5.2; 63% of tumors were organ confined. In univariate analyses 8q gain (odds ratio OR=8.9, p

324 Background: Dasatinib (D) is an oral tyrosine kinase inhibitor of Src mediated signaling, and has demonstrated promising preclinical anti-tumor activity in bladder cancer with activated Src (Sonpavde G, Mol Cancer Ther, 2010). We previously reported the feasibilty of neoadjuvant D before RC for MIBC in a phase II trial (Hahn NM, ASCO, 2012). Here, we report correlative tumor tissue studies. Methods: A phase II trial (N=25) was conducted to assess the safety and biologic activity of neoadjuvant D in cT2-T4a, N0, M0 bladder cancer patients (pts) unsuitable for or willing to forego platinum-based neoadjuvant therapy. Patients received D 100 mg po once daily for 28 +/- 7 days followed by RC 8-24 hours after the last dose. Correlative studies evaluated tissues from baseline biopsy and RC for expression of phosphorylated (p) SRC family kinases (SFK) by immunohistochemistry (IHC). pSFK expression was scored on a scale from 0 - 3 by 2 pathologists. Results: The study completed accrual with enrollment of 25 patients. Both baseline and RC tumor were available and adequate for correlative studies in 14 cases. In 5 additional cases, carcinoma was available for baseline or RC tumor only. All pre- and post-treatment tissue samples demonstrated positive staining by IHC in inflammatory cells, which were used as the internal positive control. In all cases, the cancer cells were positive before treatment and the staining decreased after treatment. In 6 cases there was no detectable staining after treatment. In 9 cases (66%), pSFK levels decreased by 2 units from 2.5-3 pre-treatment to 0.5-1 post-treatment. In 5 cases, levels declined

85 Background: Active surveillance (AS) allows patients with localized prostate cancer to delay and even avoid treatments that carry risk of significant side-effects. However, biomarkers are needed to more accurately risk-stratify patients and enhance acceptance of AS. Challenges with biomarkers measured from needle biopsy specimen that need to be address include limited material, tumor and specimen heterogeneity and fragmentation of RNA. Methods: A total of 23 patients with matched formalin-fixed paraffin-embedded (FFPE) biopsy and radical prostatectomy (RP) specimens were identified at University of California, San Francisco and Cedar Sinai. For each patient, samples were taken from tumor, non-neoplastic tissue adjacent to tumor (NAT) and non-neoplastic tissue contralateral to tumor (NCT) from both biopsy and RP specimens. RNA expression was measured in 130 samples using the 1.4 million feature Affymetrix Human Exon 1.0 ST arrays. Results: From 1 mm cylindrical cores punched from FFPE blocks, sufficient RNA was extracted for the assay from 63 out of 69 (91%) biopsy specimens and 69 out of 69 (100%) RP specimens. Median RNA yield from biopsies was lower than RP specimens but a similar quantity and quality of cDNA was amplified from the 100 ng RNA required for the assay. RNA from 62/63 (98%) of the biopsy specimens and 68/69 (99%) of the RP specimens generated expression data that passed quality control. Genomic classifiers (GC) predicting metastasis (GC1) and presence of high Gleason grade tumor (GC2) were highly correlated between matched biopsy and RP specimens with R=0.74 (p=0.0003) and R=0.63 (p=0.004), respectively. For GC2, matched tumor and NAT from RP had a higher correlation (R=0.57, p=0.0045) than matched tumor and NCT (R=0.10, p=0.67). There was no relationship between percentage of stromal contamination in the biopsy and the GC scores for either classifier, R=0.02 (p=0.91) and R=-0.16 (p=0.28) respectively. Conclusions: RNA expression levels can be measured in FFPE needle biopsy on a genome-wide scale with similar data quality compared to RP specimens. GC scores from biopsies and RP samples correlate well, are robust to stromal contamination, and are present in adjacent non-neoplastic tissue.

229 Background: Chronic pancreatitis (CP) and smoking are two major risk factors for pancreatic cancer (PaCa). Here we developed mouse models of PaCa by exposing mice to CP and/or smoking. Methods: Wild type (WT) and Pdx1-Cre;LSL-Kras transgenic mice were subjected to 7-hourly cerulein injections (50µg/kg) twice a week for 3 weeks to induce CP. Another group of mice was exposed to tobacco smoke (80mg/m3) in chambers for 5days/week during 6 weeks. A third group was exposed to both treatments. Mice were then sacrificed and pancreatic tissue analyzed. Pancreatic lesions, proliferation, fibrosis, inflammation, EMT and stem cell markers were analyzed by immunohistochemistry and Western. Results: CP significantly increased the number and stage of pancreatic neoplastic (PanIN) lesions with fibrosis, stellate cell activation and inflammation in transgenic mice. These changes were significantly less in WT mice. EMT markers (decrease in E-Cadherin and increase in vimentin protein levels) were stimulated by CP in both WT and transgenic mice. Similarly, smoking stimulated PanIN lesion formation, fibrosis and inflammation in transgenic mice, but to a lesser extent than CP model. Expression of EMT and stem cell markers was greater in the smoking model compared to CP. Combination of CP and smoking induced greater stimulation of PanIN lesion formation, fibrosis and inflammation compared to either treatment alone in transgenic mice. Conclusions: The results indicate that both CP and tobacco smoke promote PaCa progression in transgenic mice. The combination of CP and smoking has greater effects than either treatment alone. EMT and stem cell markers developed in WT as well as in transgenic mice with smoking suggesting that smoking has effects independent of Kras on cell transformation.

Prostate cancer (PCa) remains a principal cause of mortality in developed countries. Because no clinical interventions overcome resistance to androgen ablation therapy, management of castration resistance and metastatic disease remains largely untreatable. Metastasis is a multistep process in which tumor cells lose cell-cell contacts, egress from the primary tumor, intravasate, survive shear stress within the vasculature and extravasate into tissues to colonize ectopic sites. Tumor cells reestablish migratory behaviors employed during nonneoplastic processes such as embryonic development, leukocyte trafficking and wound healing. While mesenchymal motility is an established paradigm of dissemination, an alternate, 'amoeboid' phenotype is increasingly appreciated as relevant to human cancer. Here we discuss characteristics and pathways underlying the phenotype, and highlight our findings that the cytoskeletal regulator DIAPH3 governs the mesenchymal-amoeboid transition. We also describe our identification of a new class of tumor-derived microvesicles, large oncosomes, produced by amoeboid cells and with potential clinical utility in prostate and other cancers.

Neutral endopeptidase 24.11 (NEP, CD10) is a cell-surface enzyme expressed by prostatic epithelial cells that cleaves and inactivates neuropeptides implicated in the growth of androgen-independent prostate cancer (PC). NEP substrates such as bombesin and endothelin-1 induce cell migration. We investigated the mechanisms of NEP regulation of cell migration in PC cells, including regulation of phosphorylation on tyrosine of focal adhesion kinase (FAK). Western analyses and cell migration assays revealed an inverse correlation between NEP expression and the levels of FAK phosphorylation and cell migration in PC cell lines. Constitutively expressed NEP, recombinant NEP, and induced NEP expression using a tetracycline-repressive expression system inhibited bombesin- and endothelin-1-stimulated FAK phosphorylation and cell migration. This results from NEP-induced inhibition of neuropeptide-stimulated association of FAK with cSrc protein. Expression of a mutated catalytically inactive NEP protein also resulted in partial inhibition of FAK phosphorylation and cell migration. Coimmunoprecipitation experiments show that NEP associates with tyrosine-phosphorylated Lyn kinase, which then binds the p85 subunit of phosphatidylinositol 3-kinase (PI3-K) resulting in an NEP-Lyn-PI3-K protein complex. This complex competitively blocks FAK-PI3-K interaction, suggesting that NEP protein inhibits cell migration via a protein-protein interaction independent of its catalytic function. These experiments demonstrate that NEP can inhibit FAK phosphorylation on tyrosine and PC cell migration through multiple pathways and suggest that cell migration which contributes to invasion and metastases in PC cells can be regulated by NEP.

The scope of this grant proposal is to investigate whether adhesion of prostate cancer cells to extracellular matrices regulates cell growth and survival. We have shown that attachment of LNCaP cells to a variety of matrices results in the phosphorylation of the focal adhesion kinase(FAK) and the scaffolding protein, p130. Downstream effectors of this signalling pathway in LNCaP cells include the Crk family of proteins, and the Crk binding proteins, C3G and DOCK180. Therefore upon adhesion of LNCaP cells, signalling pathways to the nucleus and cytoskeleton become activated to cause cell proliferation and cytoskeletal reorganization. We have set up a proliferation assay and measured a 15 - 20% increase in cell proliferation upon adhesion of LNCaP cells to fibronectin, collagen I and matrices generated by cultured osteoblastic cells. In addition, adhesion also prolonged the survival of LNCaP cells. Our results demonstrate, that adhesion to the substratum plays an important role in the regulation of proliferation and survival of LNCaP cells.

Clinicians have long been hampered by the inability to distinguish patients with localized prostate cancer who will and will not respond to radiotherapy. In a significant proportion of patients therapy fails as determined by increasing posttreatment serum prostate specific antigen (PSA). We evaluated the expression of 2 key regulators of apoptosis, bcl-2 and p53, relative to treatment outcomes in patients who received external beam radiotherapy for clinically organ confined carcinoma of the prostate.Immunohistochemical staining for bcl-2 and p53 on pretreatment needle biopsies was performed in 54 patients who were treated with radiotherapy for localized prostate cancer. Expression was scored using strict criteria. Nadir PSA less than 1 ng./ml. after therapy was considered a successful treatment response.There was a predominance of stage T1c cancer (74%) with a mean Gleason score of 6.9 and an average pretreatment PSA of 25.3 ng./ml. Overall 54% of the patients did not have a nadir PSA of less than 1 ng./ml. Of the bcl-2 positive cases therapy ultimately failed in 85%. Similarly 88% of the patients with p53 positive biopsies had treatment failure and in all with bcl-2 as well as p53 expression radiotherapy failed. Expression of bcl-2 and p53 was an independent prognostic variable for treatment failure with odds ratios (95% confidence interval) of 7.3 and 10.8, respectively.Expression of bcl-2 and p53 was associated with treatment failure after external beam radiation therapy. These findings suggest that bcl-2 and p53 expression in pretreatment biopsies may be helpful for predicting response to definitive radiotherapy.

Approximately 30% of clinically localized prostate adenocarcinomas treated by radical prostatectomy (RP) will recur within 10 years. To prevent recurrence, new adjuvant therapies are in development that seek to treat high-risk patients after surgery. To identify patients as candidates for these treatments, improved biomarkers for predicting prognosis are needed. Reduced expression of E-cadherin has been proposed as a new marker for predicting prognosis in prostate adenocarcinoma. Since few studies have examined the relation between risk factors for disease progression and E-cadherin expression using routinely processed RP specimens, we used RP specimens to correlate downregulation of E-cadherin and pathologic stage at RP.Primary adenocarcinomas (n = 76) from formalin-fixed and paraffin-embedded RP specimens were evaluated by immunohistochemistry against E-cadherin (HECD-1) using heat-induced epitope retrieval and automated staining (BioTek Solutions). Normal appearing prostate epithelium was used as an internal control for each specimen. Staining was scored as an estimate of the percentage of tumor cells in each specimen that showed strong plasma membrane staining.Specimens were divided into three categories with respect to Gleason score: intermediate (score 5 to 6, n = 31), intermediate to high (score 7, n = 25), and high (score 8 to 9, n = 20). For pathologic stage, specimens were divided into three categories: low stage/organ confined (pT2, n = 30), intermediate stage/limited extraprostatic extension (pT3a, n = 25), and high stage/seminal vesicle-pelvic lymph node metastases (pT3b-any pTN1, n = 21). In univariate analysis, reduced levels of E-cadherin correlated with advanced Gleason score (P = 0.003) and advanced pathologic stage (P = 0.008). In multivariate analysis, E-cadherin, preoperative prostate-specific antigen, and Gleason score all contributed independently to the prediction of high-stage disease (P0.0001). Ten pelvic lymph node metastases from this same patient cohort were stained for E-cadherin. All were positive and 9 of 10 were moderately to strongly positive.Since essentially all patients in the high-stage category have a very high likelihood of disease recurrence, we conclude that the study of E-cadherin in a prospective manner as a potential biomarker of disease progression in patients with clinically organ-confined prostate cancer who undergo RP is warranted. Additionally, our finding that most metastatic tumor cells in pelvic lymph nodes express E-cadherin supports the notion that the establishment of the distant colonization and growth of metastatic tumor cells may be facilitated by expression or re-expression of previously downregulated E-cadherin. This would strongly suggest that irreversible genetic inactivation through mutation or allelic loss at 16q2.3 is probably not the mechanism of E-cadherin downregulation in most abnormally expressing primary prostate carcinomas.

Many Src Homology 3 (SH3) domains function as molecular adhesives in intracellular signal transduction. Based on previous ultrastructural studies, short motifs which bind to the first SH3 domains of the adapters Crk and CRKL were selectively mutagenised to generate Crk/CRKL SH3-binding peptides of very high affinity and selectivity. Affinities were increased up to 20-fold compared to the best wildtype sequences, while the selectivity against a similar SH3 domain [Grb2SH3(N)] was not only retained, but sometimes increased. Blot techniques with GST-fusion peptides and in solution precipitation assays with biotinylated high affinity Crk binding peptides (HACBPs) were subsequently used to analyse the binding of these sequences to a large panel of SH3 domain-containing fusion proteins. Only those proteins which contained the CrkSH3(1) or CRKLSH3(1) domains bound efficiently to the HACBPs. A GST-HACBP fusion protein precipitated Crk and CRKL proteins out of 35S-labelled and unlabelled cell lysates. Very little binding of other cellular proteins to HACBP was detectable, indicative of a great preference for Crk and CRKL when compared to the wide variety of other endogenous cellular proteins. Moreover, HACBP disrupted in vitro preexisting Crk-complexes with DOCK180 and the exchange factors SoS and C3G, which are known targets of Crk adapters, in a concentration dependent manner. HACBP-based molecules should therefore be useful as highly selective inhibitors of intracellular signalling processes involving Crk and CRKL.

MicroRNAs (miRNAs) are small (∼22 nucleotide) non-coding RNAs that regulate a myriad of biological processes and are frequently dysregulated in cancer. Cancer-associated microRNAs have been detected in serum and plasma and hold promise as minimally invasive cancer biomarkers, potentially for assessing disease characteristics in patients with metastatic disease that is difficult to biopsy. Here we used miRNA profiling to identify cancer-associated miRNAs that are differentially expressed in sera from patients with metastatic castration resistant prostate cancer (mCRPC) as compared to healthy controls. Of 365 miRNAs profiled, we identified five serum miRNAs (miR-141, miR-200a, miR-200c, miR-210 and miR-375) that were elevated in cases compared to controls across two independent cohorts. One of these, miR-210, is a known transcriptional target of the hypoxia-responsive HIF-1α signaling pathway. Exposure of cultured prostate cancer cells to hypoxia led to induction of miR-210 and its release into the extracellular environment. Moreover, we found that serum miR-210 levels varied widely amongst mCRPC patients undergoing therapy, and correlated with treatment response as assessed by change in PSA. Our results suggest that (i) cancer-associated hypoxia is a frequent, previously under-appreciated characteristic of mCRPC, and (ii) serum miR-210 may be further developed as a predictive biomarker in patients with this distinct disease biology.

The Crk protein belongs to the family of proteins consisting of mainly Src homology 2 and 3 (SH2 and SH3) domains. These proteins are thought to transduce signals from tyrosine kinases to downstream effectors. In order to understand the specificity and effector function of the SH3 domain of Crk, we screened an expression library for binding proteins. We isolated Eps15, a substrate of the epidermal growth factor receptor (EGFR) tyrosine kinase, and Eps15R, a novel protein with high sequence homology to the carboxyl-terminal domain of Eps15. Antibodies raised against a fragment of the Eps15R gene product immunoprecipitated a protein of 145 kDa. Eps15 and Eps15R bound specifically to the amino-terminal SH3 domain of Crk and coprecipitated equivalently with both c-Crk and v-Crk from cell lysates. The amino acid sequences of Eps15 and Eps15R featured several proline-rich regions as putative binding motifs for SH3 domains. In both Eps15 and Eps15R, we identified one proline-rich motif which accounts for their interaction with the Crk SH3 domain. Each binding motif contains the sequence P-X-L-P-X-K, an amino acid stretch that is highly conserved in all proteins known to interact specifically with the first SH3 domain of Crk. Furthermore, we found that immunoprecipitates of activated EGFR-kinase stably bound in vitro-translated Eps15 only in the presence of in vitro-translated v-Crk. Crk might therefore be involved in Eps15-mediated signal transduction through the EGFR.

Integrin cell adhesion receptors are internalized and recycled to the plasma membrane during cellular migration. The laminin binding A3B1 and A6B1 integrin receptors are differentially expressed on prostate tumor cells in Gleason grades 3-5 as detected in biopsy and prostatectomy specimens. The A6 integrin exists as a full length form or as a pro-metastatic variant, A6pB1, a receptor form missing the extracellular laminin binding domain via the action of the serine protease, uPA. The objective of this study is to determine whether internalization of the A6 integrin occurs in human bone metastatic lesions and whether cross-talk between the laminin receptors A6B1, A6pB1 and A3B1 modifies their respective internalization rates. In a tissue microarray of 185 metastatic prostate cancers from 45 patients, approximately 70% of bone metastases expressed A6B1 on tumor plasma membrane and/or within the cytoplasm. The A6 and A6p integrins in DU145 cells are constitutively internalized with a biological half life of approximately 30 minutes as compared to 60 minutes for A3 or 20 minutes for the unrelated transferrin receptor. Increasing production of A6pB1 by supplying exogenous uPA stimulated A3 internalization. Conversely, blockage of A6pB1 production using an A6 specific antibody (J8H) decreased A3 internalization. Targeted depletion of the A3 integrin increased DU145 cell migration that was dependent upon A6 integrin as detected by antibody blocking experiments using J8H (A6 specific) and AIIB2 (B1 specific). Analysis of transcriptomic data from prostate cancer xenografts, revealed three different candidate receptor recycling genes that significantly correlated with the loss of membrane and increase in cytoplasmic expression. Taken together, these data indicate that cross-talk between A6B1 and A3B1 modifies their internalization rates and prostate tumor cell migration. This suggests that successful therapeutic targeting of metastasizing prostate cancer cells may depend upon coordinated targeting of both receptors and the recycling machinery. Current work is to determine the involvement of candidate recycling genes in the biological control of laminin binding integrins in human prostate cancer. (Supported in part by NIH grants P30CA023074, 1RO1 CA159406 and the TACMASS core service of the University of Arizona Cancer Center). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2457. doi:1538-7445.AM2012-2457

Introduction: Prostate cancer (PCa) metastasizes to a number of sites, however, it has a propensity to metastasize to bone. There is considerable variation in sites of metastases, the phenotype of metastases, and bone response in PCa. Our objective was to detail the behavior of PCa metastases in a large cohort of patients who have died of PCa. Methods: Tissue samples used in this study were obtained from seventy patients who had died from advanced PCa and who underwent a rapid autopsy, which was performed under the aegis of the Prostate Cancer Donor Program at the University of Washington. We evaluated the clinical history, sites of metastasis, pathology, biomarker expression and bone response for all 70 patients. We used laser capture microdissection to isolate mRNA from liver, lymph node and bone metastases and performed expression analyses on AgilentTM Oligo Arrays (n=32). Tissue microarrays were used to determine the number of neuroendocrine metastases, androgen receptor positivity, androgen-associated protein expression, and PCa proliferation rates (Ki-67) from 42 patients. Histomorphometry was used to evaluate bone response in ∼20 predetermined bone biopsy sites from 50 of the patients. Results: PCa bone metastases occurred in 90% of our patients that died of PCa. Additionally, the majority of bone metastases were predominantly osteoblastic 71%, with 20% predominantly osteolytic and 9% with a mixed or no bone response. The predominant non-bone sites were lymph nodes, liver, lung and adrenal glands respectively. Seven of 42 patients had metastases with a neuroendocrine phenotype. Nuclear androgen receptor expression was high in bone and low in liver metastases and the correlation between nuclear AR and cytoplasmic PSA was 0.49. Proliferation rates were low in normal prostate increasing in Gleason 3 through 4, and higher in metastases. In a preliminary expression analysis we observed significantly higher expression of CD302, ZNF329, and TMEM79 in the PCa bone metastases compared to soft tissue metastases. Interestingly, bone histomorphometry revealed that the number of TRAcP positive osteoclasts was halved in bone cores from patients treated with bisphosphonates (n=459) compared to bone cores from non-bisphosphonate treated patients (n=284) (p=0.0003). Conclusions: The heterogeneity of PCa metastases both within and between patients is a considerable obstacle in determining the appropriate treatment regime for each individual. Our data highlight these differences, while suggesting patients may be grouped into cohorts with consistent molecular signatures and clinical outcomes. 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 3302.

Cloning and sequencing of chicken C-terminal Src kinase (CSK), a tyrosine kinase that phosphorylates the regulatory C-terminal tyrosine residue present on cytoplasmic tyrosine kinases of the Src family, demonstrated a high degree of interspecies conservation as well as src homology 2 and 3 domains N-terminal to the kinase domain. The lack of autophosphorylation sites distinguishes CSK from other tyrosine kinases. CSK is unique and does not belong to a gene family, suggesting that it may phosphorylate other members of the Src family of tyrosine kinases in addition to c-Src. Since complex formation between c-Src and CSK seemed a likely regulatory step in the control of c-Src kinase activity, such an association was investigated by immunoprecipitation and Western blotting as well as intracellular localization studies. Although some portions of CSK were found in a membrane fraction, no complex formation between CSK and c-Src was observed, suggesting that the src homology 2 domain of CSK does not play a role in the direct interaction of c-Src.

We have previously demonstrated that plasminogen immobilized on various surfaces forms a substrate for efficient conversion to plasmin by tissue plasminogen activator (t-PA) (Silverstein, R. L., Nachman, R. L., Leung, L. L. K., and Harpel, R. C. (1985) J. Biol. Chem. 260, 10346-10352). We now report the binding of human plasminogen to the extracellular matrix synthesized in vitro by cultured endothelial cell monolayers. The binding was specific, saturable at plasma plasminogen concentrations, reversible, and lysine-binding site-dependent. Functional studies demonstrated that matrix immobilized plasminogen was a much better substrate for t-PA than was fluid phase plasminogen as shown by a 100-fold decrease in Km. Activation of plasminogen by t-PA and urokinase on the matrix was equally efficient. The plasmin generated on the matrix, in marked contrast to fluid phase, was protected from its fast-acting inhibitor, alpha 2-plasmin inhibitor. Matrix-associated plasmin converted bound Glu- into Lys-plasminogen, which in turn is more rapidly activated to plasmin by t-PA. The extracellular matrix not only binds and localizes plasminogen but also improves plasminogen activation kinetics and prolongs plasmin activity in the subendothelial microenvironment.

We have previously demonstrated that plasminogen activator inhibitor (PAI-1) is associated with the extracellular matrix of cultured bovine smooth muscle cells (Knudsen, B.S., Harpel, P.C., Nachman, R.L. (1987) J. Clin. Invest. 80, 1082-1089). In this report we describe the physiologic role of PAI-1 during the interaction of the tissue plasminogen activator (t-PA) secreting Bowes human melanoma cell line with endothelial extracellular matrices. In addition we have characterized the t-PA.PAI complexes formed during this interaction in the presence and absence of plasminogen. In the absence of plasminogen, a 104-kDa complex between Bowes t-PA and PAI-1 appears in the supernatant. In the presence of plasminogen, PAI initially prevents plasmin formation on the matrix and protects the matrix from degradation by plasmin. The 104-kDa t-PA.PAI complex is degraded into a 68 and a 47-kDa complex by small amounts of plasmin generated from secreted Bowes t-PA and plasminogen. Analysis of these complexes revealed that t-PA is rapidly cleaved by plasmin within the complex whereas complexed PAI-1 is not further degraded. Matrix-associated PAI-1 may play an important role in the protection of extracellular matrices from remodeling and degradation by cellular t-PA and plasminogen.

The extracellular matrix secreted by cultured bovine smooth muscle cells (BSMC) contains an endothelial type plasminogen activator (PA) inhibitor. When PA is incubated with the matrix, a high molecular weight complex containing a truncated PA inhibitor is released into the supernatant. The inhibitor also dissociates from the matrix by treatment with glycine, pH 2.7, in its intact, functionally active, 45-kD form, whereas treatment of the matrix with thrombin results in the release of a cleaved, inactive, 41 kD PA inhibitor. Bowes melanoma cells but not smooth muscle cells cultured on BSMC matrices decrease available matrix associated PA inhibitor. PA inhibitor incorporated into the extracellular matrix may serve an important role in the regulation of plasminogen activator mediated matrix degradation.

The specificity of SH3 domain complex formation plays an important role in determining signal transduction events. We have previously identified a highly specific interaction between the first CrkSH3 domain [CrkSH3(1)] and proline-rich sequences in the guanine nucleotide exchange factor C3G. A 10 amino acid peptide derived from the first proline-rich sequence (P3P4P5A6L7P8P9K10K11R12) bound with a Kd of 1.89 +/- 0.06 microM and fully retained the high affinity and unique selectivity for the CrkSH3(1) domain. Mutational analysis showed that P5, P8, L7 and K10 are critical for high affinity binding. A conservative mutation, K10R, significantly decreased the affinity for the CrkSH3(1) domain while increasing the affinity for Grb2. Comparative binding studies with the K10R and K10A mutant peptides to c-Crk and v-Crk further suggested that K10 binds via a charge-dependent and a charge-independent interaction to the RT loop of the CrkSH3(1) domain. Besides determining important structural features necessary for high affinity and specificity binding to the CrkSH3(1) domain, our results also demonstrate that a conservative mutation in a single amino acid can significantly alter the specificity of an SH3 binding peptide.