Supplementary Figure 1 from The Cell Fate Determination Factor Dachshund Inhibits Androgen Receptor Signaling and Prostate Cancer Cellular Growth

Background Cystic fibrosis (CF) is the most commonly inherited lethal autosomal recessive genetic disease amongst Caucasians. CF results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Patients with homozygous or compound heterozygous CFTR mutations have a risk of pancreatitis, but typically do not live long enough to develop pancreatic ductal adenocarcinoma (PDA), a disease that has an average age at diagnosis of 65 years. Little is known about the risk of the development of PDA in people who are heterozygous for mutations in the CFTR gene.Patients and Methods We report a case of a patient with PDA who underwent resection, who is a carrier for the W1282X nonsense mutation in the CFTR gene. The patient is of Ashkenazi Jewish ethnicity and has a family history of CF, but no family history of PDA. We reviewed the English language literature for the prevalence of PDA in CF patients (and CFTR mutations in the setting of PDA) and their significance in terms of screening, and the use of this mutation as a biomarker for an increased risk of the development of PDA.Conclusion We conclude that patients with CFTR mutations, who also have other risks for the development of PDA such as a family history of the disease, should undergo screening and be educated about their risks.

significantly lower than Control Group (p<0.05).A significant reduction on liver MDA content and on mitochondrial dysfunction were observed in Diazoxide Group compared to Control Group (p<0.05).No differences in pulmonary vascular permeability and MPO activity were observed between groups.Twenty four hours after reperfusion Diazoxide Group showed a reduction of AST and ALT serum levels when compared to Control group (p<0.05).Conclusion: Diazoxide attenuates liver ischemia/reperfusion injury probably by a mechanism related to mitochondrial function preservation during liver ischemia.

Signal transducer and activator of transcription-3 (Stat3) is frequently activated in breast cancer and multi- ple lines of evidence suggest that Stat3 promotes tumor progression. However, the prognostic value of Stat3 in hu- man breast cancer remains controversial and associations range from favorable to unfavorable based on four out- come studies of 62, 102, 255 and 517 patients. Cellular Stat3 protein expression was measured in three studies whereas nuclear localized, tyrosine phosphorylated Stat3 (Nuc-pYStat3) was used as the readout in only one study. We therefore retrospectively analyzed the prognostic value of Nuc-pYStat3 in a larger material of 721 breast cancer specimens. Overall, patients whose tumors were positive for Nuc-pYStat3 tended to have improved survival, but the trend did not reach statistical significance (P=0.08). When specimens were stratified by tumor grade, patients with low grade but not high grade tumors that were positive for Nuc-pYStat3 had significantly prolonged overall survival in univariate analysis (P=0.014) but not in multivariate analyses. Unexpectedly, quantitative immunofluoresence detec- tion revealed highest levels of Nuc-pYStat3 in normal breast epithelia and gradual loss of Nuc-pYStat3 during pro- gression from DCIS, invasive ductal carcinoma, and lymph node metastases. Levels of Nuc-pYStat3 correlated posi- tively with levels of Nuc-pYStat5, a favorable prognostic marker, in invasive ductal carcinomas. Furthermore, Nuc- pYStat3 levels correlated strongly with protein levels of nuclear localized Stat5a (r=0.633, P<0.001) but not Stat5b. Our data does not support the notion that Nuc-pYStat3 is an independent marker of prognosis in breast cancer, al- though future studies may reveal prognostic utility within molecularly characterized subtypes of breast cancer.

Synovial sarcomas are rare tumors which most often present in the distal extremities of young adults and children. There are unique clinicopathologic features of this tumor which lend themselves to multidiscipli- nary treatment and translational research. We present a case of a primarily cystic synovial sarcoma and pro- vide a thorough review of the available literature.

Abstract Despite the routine use of chemotherapy and radiotherapy, survival has not significantly improved in patients with pancreatic ductal adenocarcinoma (PDA), a situation that signifies the urgent need for novel therapeutic approaches. Angiotensin II (AngII), the principal hormone of the renin angiotensin system, is actively generated in the pancreas and has been suggested as a key mediator of PDA cell survival. Fatty acid synthase (FAS) is a key enzyme involved in synthesis of fatty acids and its high levels have been correlated with poor prognosis in many cancers. In this study, we investigated the potential molecular basis for the role of AngII in pancreatic carcinogenesis through studying its effect on FAS. AngII significantly increased the expression of FAS mRNA and protein in Panc-1 and PK9 PDA cells lines and induced FAS promoter activity. AngII-FAS mRNA induction was inhibited by an AngII type 1 receptor (AT1R) antagonist, losartan, and to a lesser extent by an AT2R antagonist. AngII activated the phosphorylation of ERK1/2, but not p38 or c-Jun NH2-terminal MAP kinases. Inhibition of ERK1/2 activation reduced the AngII-induced FAS synthesis. AngII activated the phosphorylation of AKT. Inhibition of AKT activation prevented the AngII-mediated increase of FAS. In human premalignant (n=6) and invasive PDA lesions (n=25), high expression levels of FAS correlated well with tumor stage and invasion status, and with high expression levels of angiotensin converting enzyme (ACE). Immunohistochemical staining of PDA serial sections show co localization of ACE with FAS in the malignant ducts and stromal cells. Oral administration of losartan significantly (p< 0.05) decreased the growth of PANC-1 and PK-9 cells xenografted on the flank of nude mice and significantly (p<0.05) reduced the expression of FAS mRNA and protein in the xenografts. Our data suggest a positive autocrine/paracrine action for the local pancreatic AngII generating system during pancreatic carcinogenesis, and provide the first insight into an AngII-initiated signal transduction pathway that regulates fatty acid metabolism and modulate lipogenesis in PDA. These results raise the possibility that AngII blockade therapies could be potential candidates in novel treatment strategies of PDA. 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 51.

CONTEXTMalignant melanoma commonly metastasizes to the small intestine where it can cause pain, bleeding, and obstruction. However, jaundice from metastatic melanoma is relatively uncommon.CASE REPORTA case of known malignant melanoma presenting as new onset obstructive jaundice as a result of a rarely reported metastasis to the ampulla of Vater.CONCLUSIONMultidisciplinary management of patients with metastatic melanoma is essential.

Background. Cigarette smoke and nicotine are among the leading environmental risk factors for developing pancreatic ductal adenocarcinoma (PDA) We showed recently that nicotine induces osteopontin (OPN), a protein that plays critical roles in inflammation and tumor metastasis. We identified an OPN isoform, OPNc, that is selectively inducible by nicotine and highly expressed in PDA tissue from smokers. In this study, we explored the potential proinflammatory role of nicotine in PDA through studying its effect on the expression of monocyte chemoattractant protein (MCP)-I and evaluated the role of OPN in mediating these effects.Methods. MCP-1 mRNA and protein in PDA cells treated with or without nicotine (3-300 nmol/L.) or OPN (0 15-15 nmol/L) were analyzed by polymerase chain reaction and enzyme-linked immunosorbent assay Luciferase-labeled promoter studies evaluated the effects of nicotine and OPN on MCP-I transcription Intracellular and tissue colocalization of OPN and MCP-I were examined by immunofluorescence and immunohistochemistry.Results. Nicotine treatment significantly increased MCP-1 expression IN PDA cells Interestingly, blocking OPN with siRNA or OPN antibody abolished these effects. Transient transfection of the OPNc gene, in PDA cells or their treatment with recombinant OPN protein significantly (P < .05) increased MCP-1 mRNA and protein and induced its promoter activity MCP-I was found in 60% of invasive PDA lesions, of whom 66% were smokers. MCP-1 colocalized with OPN in PDA cells and in the malignant ducts, and correlated well unlit higher expression levels of OPN in the tissue from patients with invasive PDAConclusion. Our data suggest that cigarette smoking and nicotine may contribute to PDA nil lamination by inducing MCP-I and provide a novel insight into a unique role for OPN in mediating these effects. (Surgery 2010,148 298-309.)

Recent preclinical data have demonstrated that pancreatic adenocarcinoma (PDA) cells with defects in the Fanconi anemia/BRCA2 pathway are hypersensitive to interstrand crosslinking agents. The challenge is to efficiently identify patients who will benefit from these therapies. Patients were chosen for this study by evaluating personal history, ethnic background and family history of pancreatic malignancy. Molecular assays were performed on tissue samples. Patient A developed PDA in the context of a known BRCA2 frameshift mutation (2157delG), suspected because of her personal and multigenerational family history of breast cancer. She was treated with surgical resection, and targeted chemotherapy. Patient A continues to be disease free 32 months after her diagnosis and treatment. Patient B developed PDA in the context of a strong family history of PDA and Ashkenazi Jewish heritage. Genetic analysis on critical DNA repair genes revealed no alterations. This patient did not receive a tailored treatment regimen. This study highlights the challenge of treating PDA patients and selecting those eligible for targeted therapy. The current targeted treatment options for PDA are reviewed. A new multidisciplinary approach for stratifying PDA patients for promising targeted adjuvant therapy and familial risk counseling is proposed.

Abstract PARP-inhibitors (PARPi) are a novel class of agents that target cancer cells deficient in DNA repair. Early phase trials show promising success with PARP-inhibitors, yet drug resistance mechanisms and off-target pathways affected by drug exposure have not been explored. HuR (ELAV1) is a protein that binds and stabilizes mRNA transcripts upon certain stress stimuli. These HuR mRNA targets result in an increase in protein expression; and thus, protein function. Recently, we demonstrated that HuR binds and regulates deoxycytidine kinase, the gemcitabine metabolizing enzyme. Thus, HuR expression enhances gemcitabine efficacy against pancreatic cancer cells. We subsequently screened other chemotherapeutics that may stimulate the HuR stress response. In isogenic pancreatic cancer cell lines, stable HuR overexpression rendered cells up to 2-fold more sensitive to PARPi compared to control cells. Accordingly, siRNA knock down of HuR expression in both pancreatic and ovarian cancer cell lines caused PARPi resistance compared to a scramble-sequence control siRNA. In a pancreatic cancer cell line, MiaPaCa2, a 2.5-fold siRNA knock down of HuR mRNA expression detected by qPCR rendered cells approximately 4-fold more resistant to PARPi. Similarly, a modest siRNA knock down of HuR in an ovarian cancer cell line, A2780, rendered cells more resistant to PARPi. Immunofluorescence studies indicate that a 5 hour treatment with 75 µM PARPi directly caused HuR to transport from the nucleus to the cytoplasm, presumably moving ARE-rich mRNA cargoes after drug exposure. Focused gene expression analysis on HuR bound, immunoprecipitated RNA defined functional HuR downstream targets (including deoxycytidine kinase). Current studies will reveal and validate the importance of these downstream mRNA targets in PARP-inhibitor metabolism and efficacy. We hypothesize that HuR may be central to PARPi effectiveness and may be useful in understanding PARPi de novo drug resistance mechanisms. Future work will reveal whether HuR status can be utilized as a predictive marker for PARP-inhibitor-based therapy and if all clinically available PARP-inhibitors engage HuR's regulation of unique mRNA cargoes in cancer cells upon treatment. 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 3643.

Substantial evidence indicates that exposure to cigarette smoke is associated with an elevated risk of pancreatic ductal adenocarcinoma (PDA). However, the mechanisms underlying the effects of nicotine on the development or progression of PDA remain to be investigated. Previously, we showed that nicotine promotes the expression of osteopontin c (OPNc), an isoform of OPN protein that confers on cancer cells a migratory phenotype. In this study, we explored the potential prometastatic role of nicotine in PDA through studying its effect on the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) and evaluated the role of OPN in mediating these effects.

Background. Osteopontin (OPN) is a secreted phosphoprotein that confers on cancer cells a migratory Phenotype. We demonstrated recently that nicotine, a major risk factor in pancreatic ductal adenocarcinoma (PDA), increases OPN expression in PDA cells. An OPN splice variant, OPNc, supports anchorage independence and maybe the most potent OPN isoform to convey metastatic behavior In this study, roe tested the effect of nicotine on OPNc expression and analyzed the correlation between total OPN/OPNc levels and patients' smoking history.Methods. Real-time polymerase chain reaction and ultraviolet light illumination of ethidium-bromide staining were used to examine the mRNA expression in tissue and in PDA cells treated with or without nicotine (3-300 nmol/L). OPN and OPNc were localized by immunohistochemistry, and an enzyme-linked immunosorbent assay was used to analyze OPN serum levels.Results. Nicotine treatment of PDA cells selectively induced de novo expression of OPNc. OPNc was found in 87% of invasive PDA lesions, of which 73% were found in smokers. The levels of OPNc correlated well with higher expression levels of total OPN in the tissue and serum from patients with invasive PDA.Conclusion. Our data suggest that smoking and nicotine may contribute to PDA metastatic Potential through promoting OPNc expression. Although the direct role of OPNc in PDA progression is not defined, OPNc may have value as a diagnostic and prognostic marker, especially in invasive PDA. (Surgery 2009; 146:232-40.)

Background: Our objective is to study the characteristics of extrapancreatic neural plexus invasion by pancreatic carcinoma on MR imaging. Methods: 20 patients with both pancreatic carcinoma and extrapancreatic neural plexus invasion confirmed by pathology were recruited in this study. MR imaging was performed within 1 month before surgery. On MR images, signal intensity at the site of potential extrapancreatic neural plexus invasion, lymph nodes and tumor size were noted. The relationship of extrapancreatic neural plexus invasion to these findings was analyzed. Results: Signs of extrapancreatic neural plexus invasion were depicted on MR imaging in 80% of patients, which included streaky and strand-like signal intensity structure in fat tissue in 50% of patients and irregular masses adjacent to tumor in 30%. Signal intensity at invasion site was similar to that of pancreatic carcinoma. The frequencies of patients with vascular invasion and with lymph nodes larger than 5 mm were, respectively, 50% and 55%. Tumor diameter was 24 ± 7 mm on MR imaging. Extrapancreatic neural plexus invasion was correlated with vascular invasion (r = 0.58, P < 0.005), slightly related with lymphadenopathy (r = 0.35, 0.1 > P > 0.05), but not related with tumor size. Conclusion: MR imaging is useful to depict extrapancreatic neural plexus invasion by pancreatic carcinoma.

Abstract Initially isolated as the dominant suppressor of the mutant epidermal growth factor receptor (ellipse), the Dachshund gene plays a key role in metazoan development regulating the Retinal Determination Gene Network. Herein, the DACH1 gene was expressed in normal prostate epithelial cells with reduced expression in human prostate cancer. DACH1 inhibited prostate cancer cellular DNA synthesis, growth in colony forming assays, and blocked contact-independent growth in soft agar assays. DACH1 inhibited androgen receptor (AR) activity, requiring a conserved DS Domain (Dachshund domain conserved with Ski/Sno) that bound NCoR/HDAC and was recruited to an androgen-responsive gene promoter. DACH1 inhibited ligand-dependent activity of AR mutations identified in patients with androgen-insensitive prostate cancer. The DS domain was sufficient for repression of the AR wild-type but failed to repress an AR acetylation site point mutant. These studies show a role for the Retinal Determination Gene Network in regulating cellular growth and signaling in prostate cancer. [Cancer Res 2009;69(8):3347–55]

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO The Hu/ELAV mRNA binding protein HuR has functionally been linked to the tumorigenesis process. HuR binds U- or AU-rich sequences embedded in the 3' untranslated regions (UTRs) of target mRNAs, translocating and stabilizing transcripts in the cytoplasm. Here we surveyed for the first time the expression and cellular distribution of HuR in clinical specimens of pancreatic ductal adenocarcinomas (PDA). We observed predominantly cytoplasmic HuR in the most aggressive forms of PDAs, poorly differentiated PDAs (7 of 9 cases) and moderately differentiated PDAs (7 of 20), as compared to well-differentiated PDAs (0 of 6) and normal pancreatic tissue where HuR expression was almost exclusively nuclear. Thus, the cytoplasmic localization of HuR, which is linked to its mRNA stabilizing influence, correlated with clinicopathologic parameters including differentiation status and tumor size. Similarly, by immunofluorescence, we detected an increase in cytoplasmic HuR levels in cultured pancreatic cancer cells treated with apoptotic stimuli such as staurosporine, tamoxifen, and gemcitabine. Stable overexpression of HuR in 3 different pancreatic cancer cell lines renders them strikingly and selectively hypersensitive to gemcitabine (up to 50-fold in some instances), the reference drug used to treat pancreatic cancer patients. This hypersensitivity is accompanied with an enrichment in S-phase cells and a significant increase in apoptosis within 48 hours post treatment. We also discovered that HuR binds to a transcript that encodes the enzyme deoxycytidine kinase (dCK), which functions as the rate-limiting and activating enzyme of gemcitabine cellular metabolism. HuR is computationally predicted to bind to several segments of the dCK 3\#8217;-UTR, likely contributing to the stabilization of the dCK mRNA. Accordingly, HuR overexpression dramatically increased dCK protein expression in pancreatic cancer cells. Further confirmation that HuR sensitization to gemcitabine was elicited via dCK was obtained from experiments showing that the chemotherapeutic cytarabine (Ara-C), which also utilizes dCK as an activating enzyme, was preferentially cytotoxic in cells expressing high HuR levels. Taken together, these data support the hypothesis that HuR is a key mediator of gemcitabine efficacy in cancer cells by stabilizing the dCK mRNA. In summary, HuR-mediated stabilization of specific target transcripts such as dCK may dictate the therapeutic efficacy of gemcitabine, and thus may be a favorable target to enhance the clinical utility of this commonly used chemotherapeutic agent against multiple tumor types. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5539.

OBJECTIVE:To study the relationship of characteristics of pancreatic carcinoma on MR imaging to tumor recurrence time after surgical resection. MATERIALS AND METHODS:Twenty-seven patients with pancreatic carcinoma were followed up at least 2 years after surgical resection of the tumor. All patients had MR imaging within 1 month before surgery. The tumor's size, signal intensity, local and vascular invasion, abdominal lymphadenopathy on MR imaging and the positive surgical margin were noted. The results from MR imaging were compared with the duration after surgery until tumor recurrence and with the positive surgical margin. RESULTS:59% of patients had various degree of extrapancreatic invasion. The tumor recurrence times were, respectively, 24+/-21 months and 26+/-29 months in patients with and without vascular invasion (P=0.79). The combination of vascular with local invasion showed a correlation to the time of tumor recurrence (r=-0.34; P<0.05). Patients with positive surgical margins had a higher local invasion score on MR imaging and a shorter recurrence time than those with negative surgical margins. The number and size of lymph nodes were not related with tumor recurrence time. CONCLUSION:MR imaging was useful for predicting the recurrence of pancreatic carcinoma after surgical resection. Local invasion associated with and without vascular invasion on MR imaging was the indicator for the tumor recurrence.

Bildzukewicz, Nikolai A. MD; Costantino, Christina L.; Cozzitorto, Joseph A.; Pascal, John M. PhD; Witkiewicz, Agnes MD; Kennedy, Eugene P. MD; Yeo, Charles J. MD; Brody, Jonathan R. PhD Author Information

Pancreatic ductal adenocarcinoma is an aggressive disease. Surgical resection with negative margins (R0) offers the only opportunity for cure. Patients who have advanced disease that limits the chance for R0 surgical resection may undergo margin positive (MP) pancreaticoduodenectomy (PD), palliative surgical bypass (PB), celiac plexus neurolysis alone (PX), or neoadjuvant chemoradiation therapy in anticipation of future resection.