Strategy For Pancreatic Ductal Adenocarcinoma Research Articles

Onco-surgical Strategies in Pancreatic Ductal Adenocarcinoma

Onco-surgical Strategies in Pancreatic Ductal Adenocarcinoma

Endothelial Nitric Oxide Synthase Traffic Inducer (NOSTRIN) is a Negative Regulator of Disease Aggressiveness in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is refractory to available treatments. Delineating critical pathways, responsible for disease aggressiveness and therapeutic resistance, may identify effective therapeutic targets. We aimed to identify key pathways contributing to disease aggressiveness by comparing gene expression profiles of tumors from early-stage PDAC cases with extremely poor survival (≤7 months) and those surviving 2 years or more following surgical resection. Gene expression profiling was performed in tumors in a test cohort of PDAC (N = 50), which included short (≤7 months, N = 11) and long surviving (≥2 years, N = 14) patients, using affymetrix GeneChip Human 1.0 ST array. Key genes associated with disease aggressiveness were identified, using Cox regression, Kaplan-Meier, and pathway analyses with validations in independent cohorts for mechanistic and functional analyses. Gene expression profiling identified 1,820 differentially expressed genes between short and long survival groups with inflammatory gene network ranking first. Lower expression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) was associated with worst survival indicating its potential inhibitory role in disease progression. NOSTRIN overexpression suppressed migration and invasion of pancreatic cancer cells and enhanced sensitivity to chemotherapeutic drug gemcitabine. NOSTRIN inhibited production of nitric oxide (NO) by suppressing the activation of endothelial nitric oxide synthase (eNOS). Furthermore, miR-221, bound to the 3'UTR of NOSTRIN and suppressed its expression, and an increased miR-221 expression associated with poor survival in PDAC. Our findings showed that NOSTRIN is a potential negative regulator of disease aggressiveness, which may be targeted for designing improved treatment strategy in PDAC. Clin Cancer Res; 22(24); 5992-6001. ©2016 AACR.

Preoperative chemoradiotherapy does not compromise the feasibility of adjuvant chemotherapy for patients with pancreatic ductal adenocarcinoma.

Preoperative chemoradiotherapy (CRT) is a novel, emerging treatment strategy for pancreatic ductal adenocarcinoma (PDAC), but it remains unclear whether post-surgery adjuvant chemotherapy is feasible following preoperative CRT. This retrospective study evaluates the feasibility of adjuvant therapy after preoperative CRT. The subjects of this study were 99 consecutive patients who underwent pancreatectomy for PDAC between January, 2007 and February, 2013 in our hospital. Sixty patients received preoperative CRT: as gemcitabine (GEM) and 40Gy radiation in 28 (G-CRT group), and as GEM, S-1, and 50.4Gy radiation in 32 (GS-CRT group). We also evaluated 39 patients who underwent surgery alone (SA group). We investigated adjuvant chemotherapy induction and completion rates and the frequency of adverse events rated ≥grade 3, based on Common Terminology Criteria for Adverse Events (version 4.0) in all three groups. In the G-CRT, GS-CRT, and SA groups, the induction rates were 78% (22/28), 78% (25/32), and 72% (28/39), respectively; completion rates were 86% (19/22), 88% (22/25), and 82% (23/28), respectively; and adverse event frequencies were 36% (8/22), 28% (7/25), and 43% (12/28), respectively. No significant difference was found among the three groups. Preoperative CRT was demonstrated to be safe and did not compromise the feasibility of adjuvant chemotherapy.

Abstract 4009: The expression of PD-L1 on human and murine pancreatic ductal adenocarcinoma is enhanced by anticancer agents via the JAK/STAT pathway

Abstract Background: Pancreatic ductal adenocarcinoma(PDA) is the fourth most common cause of cancer-related death in Japan. Recently, new standard chemotherapies for PDA have been developed, but they are still largely unsatisfactory. Therefore, development of new treatment options has been required to improve the outcomes of patients with PDA. To break through this situation, blocking one of inhibitory immune checkpoints, Programmed death-1 (PD-1) /Programmed death-ligand 1 (PD-L1) pathway is considered to be a hopeful candidate for new treatment strategies for PDA. In this context, for the future combination therapy of anticancer agents and immune check point inhibitors, we investigated how anticancer agents influence the expression of PD-L1 on pancreatic cancer cell lines. Additionally we analyzed the molecular mechanism by which PD-L1 expression on the pancreatic cancer cell lines are regulated. Methods: Human PDA cell lines MIA PaCA-2, AsPC-1 and murine PDA cell line Pan02 were used in this study. These cells were adjusted to 1.0 × 105 / ml and incubated with anticancer agents (i.e. gemcitabine, paclitaxel and 5-fluorouracil) at 37°C for 24-72h. Then, the expression level of PD-L1 was determined using qRT-PCR and flow cytometry. For the blocking experiment of the JAK/STAT pathway, AG490 was used as a blocking agent. After 48h incubation of AsPC-1 cells at 37°C, cells were treated with various concentration of AG490 for 1h. Cells were stimulated with 5-fluorouracil, paclitaxel and gemcitabine, and then incubated in the presence or absence of AG490 for an additional 6-48h. The expression of PD-L1 was analyzed using qRT-PCR and flow cytometry. Stat1 and phospho-Stat1 protein were analyzed by western blotting. Results: In AsPC-1, MIA PaCA-2 and Pan02, the expression of PD-L1 was enhanced with all three anticancer agents in a concentration dependent manner. The phosphorylation of STAT1 and the increase of total STAT1 was observed in AsPC-1 when stimulated by each anticancer agents. After the treatment of JAK/STAT inhibitor, the phosphorylation of STAT1 was attenuated, and the PD-L1 upregulation induced by anticancer agents was cancelled in a concentration dependent manner. Conclusion: The stimulation of anticancer agents leads to an enhancement of PD-L1 expression on PDA cell lines. The JAK/STAT pathway is reported to be involved in IFN-γ mediated PD-L1 upregulation in lung cancer and hepatocellular carcinoma cell lines. In the present study, the phosphorylation of Stat1 and the expression of total Stat1 were enhanced after the anticancer agents treatment. Moreover, JAK/STAT inhibitor attenuated anticancer agents-induced PD-L1 expression. Taken together, the JAK/STAT pathway may be responsible for the anticancer agents mediated PD-L1 transcription. Citation Format: Toshifumi Doi, Takeshi Ishikawa, Tomoyo Yasuda, Tetsuya Okayama, Kaname Oka, Naoyuki Sakamoto, Yuji Naito, Yoshito Itoh. The expression of PD-L1 on human and murine pancreatic ductal adenocarcinoma is enhanced by anticancer agents via the JAK/STAT pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4009.

Loss of P53 Function Activates JAK2–STAT3 Signaling to Promote Pancreatic Tumor Growth, Stroma Modification, and Gemcitabine Resistance in Mice and Is Associated With Patient Survival

One treatment strategy for pancreatic ductal adenocarcinoma is to modify, rather than deplete, the tumor stroma. Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) is associated with progression of pancreatic and other solid tumors. We investigated whether loss of P53 function contributes to persistent activation of STAT3 and modification of the pancreatic tumor stroma in patients and mice. Stat3, Il6st (encodes gp130), or Trp53 were disrupted, or a mutant form of P53 (P53R172H) or transgenic sgp130 were expressed, in mice that developed pancreatic tumors resulting from expression of activated KRAS (KrasG12D, KC mice). Pancreata were collected and analyzed byimmunohistochemistry, in situ hybridization, quantitative reverse-transcription polymerase chain reaction (qPCR), or immunoblot assays; fluorescence-activated cell sorting was performed to identify immune cells. We obtained frozen pancreatic tumor specimens from patients and measured levels of phosphorylated STAT3 and P53 by immunohistochemistry; protein levels were associated with survival using Kaplan-Meier analyses. We measured levels of STAT3, P53, ligands for gp130, interleukin 6, cytokines, sonic hedgehog signaling, STAT3 phosphorylation (activation), and accumulation of reactive oxygen species in primary pancreatic cells from mice. Mice with pancreatic tumors were given gemcitabine and a Janus kinase 2 (JAK2) inhibitor; tumor growth was monitored by 3-dimensional ultrasound. STAT3 was phosphorylated constitutively in pancreatic tumor cells from KC mice with loss or mutation of P53. Tumor cells of these mice accumulated reactive oxygen species and had lower activity of the phosphatase SHP2 and prolonged phosphorylation of JAK2 compared with tumors from KC mice with functional P53. These processes did not require the gp130 receptor. Genetic disruption of Stat3 in mice, or pharmacologic inhibitors of JAK2 or STAT3 activation, reduced fibrosis and the numbers of pancreatic stellate cells in the tumor stroma and altered the types of immune cells that infiltrated tumors. Mice given a combination of gemcitabine and a JAK2 inhibitor formed smaller tumors and survived longer than mice given control agents; the tumor stroma had fewer activated pancreatic stellate cells, lower levels of periostin, and alterations in collagen production and organization. Phosphorylation of STAT3 correlated with P53 mutation and features of infiltrating immune cells in human pancreatic tumors. Patients whose tumors had lower levels of phosphorylated STAT3 and functional P53 had significantly longer survival times than patients with high levels of phosphorylated STAT3 and P53 mutation. In pancreatic tumors of mice, loss of P53 function activates JAK2-STAT3signaling, which promotes modification of the tumor stroma and tumor growth and resistance to gemcitabine. In human pancreatic tumors, STAT3 phosphorylation correlated with P53 mutation and patient survival time. Inhibitors of this pathway slow tumor growth and stroma formation, alter immune cell infiltration, and prolong survival of mice. Transcript profiling: ArrayExpress accession number: E-MTAB-3278.

Preoperative gemcitabine-based chemoradiation therapy for pancreatic ductal adenocarcinoma of the body and tail: Impact of splenic vessels involvement on operative outcome and pattern of recurrence

Among the various multimodal treatment strategies for pancreatic ductal adenocarcinoma (PDA), preoperative chemoradiation therapy (CRT) and subsequent operation is a promising strategy. The aim of this study is to evaluate the outcome of preoperative gemcitabine-based CRT for PDA of the body and tail, focusing on the associations among splenic vessel involvement, surgical outcomes, and pattern of recurrence. A total of 99 patients with PDA of the body and tail received preoperative CRT. The status of tumor involvement of the splenic artery (SA) and vein (SV) were evaluated based on radiographical findings obtained before the initiation of preoperative CRT. We assessed the following in association with the status of SA and SV involvement: (1) resection rate, (2) survival, and (3) pattern of recurrence. The resection rate of SA-positive cases (71%) was significantly lesser than that of SA-negative cases (94%; P = .004), whereas SV involvement was not associated with the resection rate. The 5-year survival rates of the resected SA-negative and SA-positive cases were 76% and 20%, respectively (P < .001). The 5-year cumulative incidence of distant recurrence was significantly higher in the SA-positive patients than in the SA-negative patients (74% vs. 17%; P < .001). In preoperative CRT for PDA of the body and tail, positive SA involvement was associated with a lesser resection rate, and the survival rate for the patients with SA-positive tumors was lesser than that for patients with SA-negative tumors because of the greater incidence of distant recurrence in SA-positive patients.

Abstract 2691: Smo-targeted sonic hedgehog signaling inhibitor enhances therapeutic antibody deposition and efficacy in a patient-derived pancreatic ductal adenocarcinoma model

Abstract Tumor stroma supports growth, invasion, and treatment resistance of pancreatic ductal adenocarcinoma (PDAC), and in conjunction with hypovascularity, constitutes a barrier that minimizes tumor cell drug exposure. Targeting tumor stroma is an attractive strategy in PDAC, and Smo-binding inhibitors are reported to mediate stromal thinning and increased microvessel density in PDAC models. No information exists as to the effect of sHHI upon deposition of macromolecular therapeutic agents such as monoclonal antibodies (mAb). Therefore we investigated effects of sHHI LDE225 upon tumor vascularity, mAb deposition, and mAb therapeutic efficacy in a patient-derived xenograft (PDX) model of PDAC in SCID mice. PDAC tumor #18269 was established in SCID mice and has retained abundant stroma and low microvessel density reminiscent of the original patient tumor. It expresses EGF receptor. Tumor-bearing mice were dosed orally with LDE225 (40 mg/kg) for 10 days, and pharmacodynamic biomarkers of sHHI effect were quantified. Mouse-specific TaqMan probes demonstrated high Gli1 expression in stromal cells, which was deeply suppressed within 3 days of sHHI dosing. Human-specific probes showed little Gli1 expression in tumor cells and no effect of sHHI treatment. Over 10 days of sHHI treatment, tumor microvessel density did not change significantly (based on CD31 staining), but the fraction of functional vessels increased significantly by day 7 (based on staining with fluorescent Lycopersicon esculentum lectin injected IV prior to tumor harvest). Fluorescently labeled mAbs that were tumor-targeted (cetuximab; anti-EGFR) or non-targeted (anti-topotecan) were co-injected on d7 of sHHI treatment. Compared to untreated controls, tumor exposure (AUC) of cetuximab more than doubled, and was observed to distribute to regions of the tumor enriched in tumor cells. Deposition of non-targeted mAb also was increased relative to controls, but distributed more to stromal areas than tumor cell areas. To investigate whether the enhanced deposition of cetuximab mediated by sHHI pretreatment would enhance efficacy, cetuximab was administered on d 5, 8, 11 of a 10-day sHHI or vehicle (control) regimen. This sequence was repeated 6 times, with an inter-cycle gap of 5 days. Rituximab (anti-human CD20) was the non-targeted control. Cetuximab alone showed no therapeutic benefit. However, cetuximab combined with sHHI suppressed tumor progression for more than 80 days, and median time to study endpoint was more than 250% longer than for all other treatment groups except sHHI-alone, which mediated a 30% increase. Conclusions: increased perfusion and vascular permeability mediated by sHHI pretreatment permits access of therapeutic mAb to otherwise inaccessible regions of the tumor interior, and reveals anti-tumor activity of a mAb that previously failed clinically for PDAC treatment. Citation Format: Jun Wang, Yang Qu, Rose Pitoniak, Sheryl A. Trueman-Fatallah, Bonnie L. Hylander, Elizabeth A. Repasky, Wen Wee Ma, Robert M. Straubinger. Smo-targeted sonic hedgehog signaling inhibitor enhances therapeutic antibody deposition and efficacy in a patient-derived pancreatic ductal adenocarcinoma 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 2691. doi:10.1158/1538-7445.AM2014-2691

Abstract C267: Sequential combination of the novel allosteric MEK1/2 inhibitor BAY 86-9766 (RDEA119) with the mTOR inhibitor rapamycin decelerates relapse of pancreatic tumors.

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) remains a devastating, deadly malignancy in the western world with a 5-year overall survival rate of less than 4% due to (1) difficult early diagnosis, (2) early metastatic spread as well as (3) high intrinsic resistance to known chemotherapies and ionizing radiation. Therefore, novel treatment strategies and improved mouse models for better prediction of clinical efficacy of new chemotherapeutic agents are urgently needed. In this study we investigated the effect of a sequential therapy strategy in a genetically engineered mouse model for PDAC combining the novel MEK1/2 inhibitor BAY 86-9766 with the mTOR inhibitor rapamycin. Experimental Procedure: Using a Cre/loxP approach we generated mice with pancreas-specific activation of oncogenic KRAS and concurrent deletion of p53 (Ptf1a+/Cre, Kras+/LSL-G12D, p53loxP/loxP; CKP) to mimic human PDAC on a molecular and morphological level. These mice typically develop lethal invasive PDAC within 8 weeks of age. Non-invasive magnetic resonance imaging (MRI) was used to track tumor progression during therapy. Primary tumor cells isolated from vehicle and BAY 86-9766 treated animals were used for molecular and functional assays to identify possible resistance mechanisms for the development of sequential therapy strategies. Results: Treatment of CKP mice with BAY 86-9766 alone resulted in an effective tumor regression already after 1 week of therapy. This massive tumor shrinkage was caused by a strong apoptosis induction specifically in tumor cells starting after 4h of treatment accompanied with caspase 3/7 activation and PARP cleavage from ex vivo samples. Moreover, BAY 86-9766 treatment resulted in a phosphorylation decrease of its target ERK1/2. Despite the potent apoptosis induction some remaining tumor cells are escaping from this induced cell death, causing the tumor relapse seen in CKP mice with continuing therapy. To investigate the underlying resistance mechanism we established long-term treated resistant cell lines. Those cells exhibit a phosphorylation upregulation of both AKT residues (thr308 and ser473) as well as of S6, a direct target of mTOR, suggesting full activation of the PI3K/AKT/mTOR signaling cascade to overcome resistance. Therefore, we started a sequential therapy strategy of BAY 86-9766 with the mTOR inhibitor rapamycin. Mice, first treated 1-2 weeks with BAY 86-9766 and afterwards with rapamycin until death, showed a significant overall survival benefit compared to their BAY 86-9766 and vehicle treated counterparts. Furthermore, we could demonstrate by MRI, that rapamycin decelerates the tumor relapse in responder mice. Conclusion: These preclinical data provide in vivo evidence, that the development of sequential therapy strategies for PDAC is of paramount importance to overcome resistance and to enhance the efficacy of targeted therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C267. Citation Format: Nicole Teichmann, Marija Trajkovic-Arsic, Arne Scholz, Roland M. Schmid, Rickmer Braren, Jens T. Siveke. Sequential combination of the novel allosteric MEK1/2 inhibitor BAY 86-9766 (RDEA119) with the mTOR inhibitor rapamycin decelerates relapse of pancreatic tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C267.

Neoadjuvant Chemotherapy with Gemcitabine and S-1 for Resectable and Borderline Pancreatic Ductal Adenocarcinoma: Results from a Prospective Multi-institutional Phase 2 Trial

Surgical resection is the only curative strategy for pancreatic ductal adenocarcinoma (PDAC), but recurrence rates are high even after purported curative resection. First-line treatment with gemcitabine and S-1 (GS) is associated with promising antitumor activity with a high response rate. The aim of this study was to assess the feasibility and efficacy of GS in the neoadjuvant setting. In a multi-institutional single-arm phase 2 study, neoadjuvant chemotherapy (NAC) with gemcitabine and S-1, repeated every 21 days, was administered for two cycles (NAC-GS) to patients with resectable and borderline PDAC. The primary end point was the 2-year survival rate. Secondary end points were feasibility, resection rate, pathological effect, recurrence-free survival, and tumor marker status. Of 36 patients enrolled, 35 were eligible for this clinical trial conducted between 2008 and 2010. The most common toxicity was neutropenia in response to 90% of the relative dose intensity. Responses to NAC included radiological tumor shrinkage (69%) and decreases in CA19-9 levels (89%). R0 resection was performed for 87% in resection, and the morbidity rate (40%) was acceptable. The 2-year survival rate of the total cohort was 45.7%. Patients who underwent resection without metastases after NAC-GS (n = 27) had an increased median overall survival (34.7 months) compared with those who did not undergo resection (P = 0.0017). NAC-GS was well tolerated and safe when used in a multi-institutional setting. The R0 resection rate and the 2-year survival rate analysis are encouraging for patients with resectable and borderline PDAC.

Cdk4/6 inhibition induces epithelial-mesenchymal transition and enhances invasiveness in pancreatic cancer cells.

Aberrant activation of Cyclin D-Cdk4/6 signaling pathway is commonly found in pancreatic ductal adenocarcinoma (PDAC). Here, we show that PD-0332991, a highly specific inhibitor for Cdk4 and Cdk6, exerted growth inhibitory effects on three human PDAC cell lines. Microarray analysis revealed that PD-0332991 downregulated cell-cycle-related genes, but upregulated genes implicated in extracellular matrix (ECM) remodeling and pancreatic cancer cell invasion and metastasis. Moreover, PD-0332991 enhanced invasion in TGF-β-responsive PDAC cell lines that harbor a wild-type SMAD4 gene (COLO-357, PANC-1), but not in TGF-β-resistant AsPC-1 cells that harbor a mutated SMAD4. PD-0332991 also induced epithelial-mesenchymal transition (EMT) in COLO-357 and PANC-1, but not in AsPC-1 cells. Inhibition of CDK4/6 using shRNA mimicked the effects of PD-0332991 on EMT induction. Furthermore, PD-0332991 increased Smad transcriptional activity in luciferase readout assays and activated TGF-β signaling. SB-505124, an inhibitor of the type-I TGF-β receptor (TβRI) kinase, completely blocked EMT induction by PD-0332991. When combined with PD-0332991, SB-505124 inhibited the growth of COLO-357 and PANC-1 cells. Taken together, these data suggest that anti-Cdk4/6 therapy could induce EMT and enhance pancreatic cancer cell invasion by activating Smad-dependent TGF-β signaling, and that combining PD-0332991 and SB-505124 may represent a novel therapeutic strategy in PDAC.

A Central Role for RAF→MEK→ERK Signaling in the Genesis of Pancreatic Ductal Adenocarcinoma

KRAS mutation is a hallmark of pancreatic ductal adenocarcinoma (PDA) but remains an intractable pharmacologic target. Consequently, defining RAS effector pathway(s) required for PDA initiation and maintenance is critical to improve treatment of this disease. Here, we show that expression of BRAF(V600E), but not PIK3CA(H1047R), in the mouse pancreas leads to pancreatic intraepithelial neoplasia (PanIN) lesions. Moreover, concomitant expression of BRAF(V600E) and TP53(R270H) result in lethal PDA. We tested pharmacologic inhibitors of RAS effectors against multiple human PDA cell lines. Mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) inhibition was highly effective both in vivo and in vitro and was synergistic with AKT inhibition in most cell lines tested. We show that RAF→MEK→ERK signaling is central to the initiation and maintenance of PDA and to rational combination strategies in this disease. These results emphasize the value of leveraging multiple complementary experimental systems to prioritize pathways for effective intervention strategies in PDA. PDA is diffi cult to treat, in large part, due to recurrent mutations in the KRAS gene. Here, we defi ne rational treatment approaches for the disease achievable today with existing drug combinations by thorough genetic and pharmacologic dissection of the major KRAS effector pathways, RAF→MEK→ERK and phosphoinositide 3′-kinase (PI3'K)→AKT.

Abstract 5635: Pegylated recombinant human hyaluronidase PH20 (PEGPH20) enhances Nab-Paclitaxel efficacy in BxPC-3 human pancreatic cancer xenografts

Abstract Hyaluronan (HA) accumulates in the extracellular matrix (ECM) of many solid tumors, including those of the prostate, colon, breast, stomach, ovary, and pancreas. This accumulation is associated with tumor progression and a negative clinical outcome. Accordingly, an HA-degrading enzyme, pegylated recombinant human hyaluronidase PH20 (PEGPH20), was developed to target tumor-associated HA in the ECM. Preclinical studies demonstrated that PEGPH20-mediated removal of HA from HA-rich xenograft tumors in mice decreased tumor interstitial fluid pressure and tumor water content resulting in a decompression of tumor vasculature, increased tumor vascular perfusion, tumor growth inhibition (TGI) and enhanced chemotherapeutic activity (Thompson 2010). We further characterized HA expression across multiple human tumor types and identified pancreatic ductal adenocarcinoma (PDA) as the cancer type with the most HA (∼87% express high levels). These observations coupled with a lack of curative therapy for PDA led us to evaluate alternative treatment strategies for PDA. Specifically, using the peritibial BxPC-3 human pancreatic cancer xenograft model, we investigated whether the antitumor activity of Nab-Paclitaxel was significantly enhanced with PEGPH20 treatment. Nude mice were inoculated with human PDA BxPC-3 cells adjacent to the right tibial periosteum, and tumor growth was monitored with ultrasonography. When tumors reached ∼400 mm3 (n≥8/group), mice were staged into 8 treatment groups: (1) vehicle control; (2) PEGPH20 monotherapy, 4.5 mg/kg; (3) Nab-Paclitaxel, 3 mg/kg; (4) Nab-Paclitaxel, 10 mg/kg; (5) Nab-Paclitaxel, 30 mg/kg; (6) Nab-Paclitaxel, 3 mg/kg, plus PEGPH20; (7) Nab-Paclitaxel, 10 mg/kg, plus PEGPH20; or (8) Nab-Paclitaxel, 30 mg/kg, plus PEGPH20. Vehicle or PEGPH20 ± Nab-Paclitaxel was administered intravenously starting on study day 0, and then dosed every third day for 15 days. At study termination, the average TGIs from animals treated with either PEGPH20 alone (12.2%), low dose (3 mg/kg) Nab-Paclitaxel (20.3%), or low dose Nab-Paclitaxel (3 mg/kg) plus PEGPH20 (25%) were not significantly different from vehicle-treated animals. However, Nab-Paclitaxel alone at both 10 mg/kg (61.5%, p&amp;lt;0.01) and 30 mg/kg (73.6%, p&amp;lt;0.01) inhibited tumor growth. High doses of Nab-Paclitaxel (30 mg/kg) ± PEGPH20 was associated with dose-related toxicity. The addition of PEGPH20 to the 10 mg/kg and 30 mg/kg Nab-Paclitaxel groups increased TGI to 71.7% (p&amp;lt;0.01), and 90.8% (p&amp;lt;0.01), respectively, relative to vehicle. These findings suggest that PEGPH20 significantly increases the anti-tumor efficacy of Nab-Paclitaxel in HA-rich PDA tumors at moderate to high Nab-Paclitaxel doses (10 and 30 mg/kg). 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 5635. doi:1538-7445.AM2012-5635

A novel 3-dimensional culture system uncovers growth stimulatory actions by TGFβ in pancreatic cancer cells

Transforming Growth Factor-β (TGF-β) exerts cell type-specific and context-dependent effects. Understanding the intrinsic effects of TGF-β on cancer cells in pancreatic ductal adenocarcinoma (PDAC) is a prerequisite for rationalized clinical implementation of TGF-β targeting therapies. Since the tumor microenvironment can affect how cancer cell respond to TGF-β, we employed a novel three-dimensional (3D) culturing system to recapitulate stromal and extracellular matrix interactions. We show here that TGF-β stimulates growth of human and murine pancreatic cancer cell lines (PCCs) when embedded in a 3% collagen IV/laminin-rich gelatinous medium (MatrigelÔ) over a solidified layer of soft agar. Moreover, in this novel 3D model, concomitant treatment with TGF-β1 and epidermal growth factor (EGF) enhanced PCC growth to a greater extent than either growth factor alone, and conferred increased chemoresistance to cytotoxic compounds. These cooperative growth-stimulatory effects were blocked by pharmacological inhibition of TGF-β type I receptor with SB431542 or the EGF receptor with erlotinib. Co-incubation with SB431542 and erlotinib enhanced the efficacy of gemcitabine and cisplatin in PCCs and in primary cell cultures established from pancreata of genetically-engineered mouse models of PDAC. These findings suggest that concomitant inhibition of TGF-β and EGF signaling may represent an effective therapeutic strategy in PDAC, and that this 3D culturing system could be utilized to test ex vivo the therapeutic response of pancreatic tumor biopsies from PDAC patients, thereby providing a functional assay to facilitate personalized targeted therapies.

Abstract 544: Blockade of CXC chemokines/CXCR2 axis in the tumor microenvironment as a potent therapeutic strategy for pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC), one of the most lethal neoplasms, is characterized histologically by expanded stromal components with marked fibrosis, which suggests that tumor-stromal interaction might play an important role in PDAC progression. We have established pancreas epithelium-specific transforming growth factor-ß receptor type II (Tgfbr2) knockout mice in the context of active Kras (KrasG12D) expression (Kras+Tgfbr2KO), which developed differentiated PDAC with 100% penetrance and recapitulated histology of human PDAC described above. In the AACR annual meetings 2009-10, we have reported that tumor-stromal interaction in the PDAC model promoted tumor progression and blockade of CXC chemokines/CXCR2 axis inhibited tumor growth and extended survival of the mice. We further examined the underlying mechanisms of tumor-stromal interaction. The CXC chemokines secreted from the PDAC cells did not change autocrine PDAC cell proliferation nor that of stromal fibroblasts, but induced CTGF expression in the stromal fibroblasts and promoted tumor angiogenesis, which resulted in promotion of tumor growth. Expression of the CXC chemokines was downregulated by TGF-β signaling, whereas, NF-κB signaling was required for the expression. Subcutaneous xenografts coinjected with the PDAC cells and fibroblasts grew faster than those with PDAC cells alone, which suggested tumor-promoting tumor-stromal interaction. We further examined the xenograft growth of CXCR2-knocked down (KD) PDAC cells and CXCR2-intact firbroblasts, compared with that of CXCR2-intact PDAC cells and CXCR2-KD fibroblasts. Results suggested that intact CXCR2 in the stromal fibroblasts was required for the tumor-promoting CXC chemokines/CXCR2 axis. In conclusion, blockade of the CXC chemokines/CXCR2 axis might have a key role in modulating the tumor microenvironment and for development of more effective therapeutic strategy for PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 544. doi:10.1158/1538-7445.AM2011-544

Abstract 5387: Combination treatment benefits of EMAP II with doxorubicin, docetaxel and gemcitabine in experimental pancreatic cancer

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer that grows rapidly, metastasizes early, and is very resistant to conventional chemotherapy. Gemcitabine, the standard first-line chemotherapeutic agent for advanced and metastatic PDAC, showed limited benefits as single agent or in combination. Endothelial monocyte activating polypeptide II (EMAP) is an antiendothelial and antiangiogenic cytokine that has been shown to enhance gemcitabine effects in PDAC. The present study evaluated the antitumor effects of combination of EMAP with doxorubicin and docetaxel in experimental PDAC. Methods: Human ASPC-1 PDAC cells and endothelial cells (HUVECs) were grown in RPMI and E-Stim medium, respectively. Cell proliferation and protein expression were analyzed by WST-1 assay and Western blotting, respectively. Apoptosis was detected by Annexin-V / propidium iodide (PI) staining. In vivo murine xenograft survival was measured after treatment with EMAP, doxorubicin, docetaxel and gemcitabine. Results: In vitro evaluation of inhibition in ASPC-1 cell proliferation showed no effect of EMAP, modest effects of doxorubicin and gemcitabine (IC50&amp;gt;10 μM) and major effects of docetaxel (IC50= 8μM); percent inhibition in cell proliferation at 10 μM concentration was 1.4, 26.5, 40 and 60, respectively. EMAP addition to doxorubicin, docetaxel or gemcitabine did not increase the effects of single agents. In HUVECs, EMAP, doxorubicin, docetaxel and gemcitabine all had significant antiproliferative effects, and percent inhibition at 10 μM concentration was 59, 79, 96 and 85, respectively. Addition of EMAP to doxorubicin, docetaxel and gemcitabine in lower concentrations (10 nM, 100 nM and 1 μM) had significant additive antiprolifertative effects. In ASPC-1 cells, no agent induced measurable apoptosis, but in HUVECs all agents either alone or in combination significantly induced apoptosis as observed by Annexin V / PI staining, PARP cleavage and phospho-JNK expression. In vivo animal survival study showed that compared to control (median survival: 21 days), EMAP (20 d) had no benefit, while doxorubicin (31 d), docetaxel (35 d) and gemcitabine (32 d) monotherapy extended overall survival. Compared to single agent therapy, combination of EMAP with docetaxel (44 d) or gemcitabine (38 d) enhanced the survival, but the combination of EMAP with doxorubicin (31 d) had no additional benefit. Conclusion: In vitro EMAP addition increased the antiproliferative and proapoptotic activity of doxorubicin, docetaxel and gemcitabine in endothelial cells but not in PDAC cells. In vivo, addition of EMAP enhanced the docetaxel and gemcitabine effect, but not that of doxorubicin, on overall animal survival. The antiendothelial combination therapy benefit through EMAP is not limited to gencitabine, and may facilitate the development of more effective therapeutic strategies for PDAC. 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 5387.

Abstract 51: Blood pressure lowering medications disrupt fatty acid metabolism in pancreatic cancer

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&amp;lt; 0.05) decreased the growth of PANC-1 and PK-9 cells xenografted on the flank of nude mice and significantly (p&amp;lt;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.

Enhanced Expression of Keratinocyte Growth Factor and Its Receptor Correlates with Venous Invasion in Pancreatic Cancer

Keratinocyte growth factor (KGF) and KGF receptor (KGFR) have been implicated in cancer growth as well as tissue development and repair. In this study, we examined whether KGF and KGFR have a role in human pancreatic ductal adenocarcinoma (PDAC). KGFR mRNA was expressed in eight pancreatic cancer cell lines, whereas the KGF mRNA was detected in seven of the cell lines and was absent in MIA PaCa-2 cells. KGFR and KGF immunoreactivity were localized in the cancer cells in 41.5 and 34.0% of patients, respectively. There was a significant correlation between KGFR or KGF immunoreactivity and venous invasion and a significant correlation between the presence of both markers and venous invasion, vascular endothelial growth factor (VEGF)-A expression, and poor prognosis. Exogenous KGF increased VEGF-A expression and release in MIA PaCa-2 cells, and PANC-1 cells stably transfected to overexpress KGF-exhibited increased VEGF-A expression. Moreover, short hairpin-KGFR transfection in MIA PaCa-2 cells reduced the stimulatory effect of exogenous KGF on VEGF-A expression. Short hairpin-KGF transfection in KLM-1 cells reduced VEGF-A expression in the cells. KGFR and KGF may act to promote venous invasion and tumor angiogenesis in PDAC, raising the possibility that they may serve as novel therapeutic targets in anti-angiogenic strategies in PDAC.

Inhibition of CDK4 impairs proliferation of pancreatic cancer cells and sensitizes towards TRAIL‐induced apoptosis via downregulation of survivin

Pancreatic ductal adenocarcinoma is one of the most common causes of cancer death in Western countries with an average survival after diagnosis of 3-6 months and a five-year survival rate under 5%. Because of the lack of effective therapies, there is the need to characterize new molecular treatment strategies. Abnormal regulation of the cell cycle is a hallmark of neoplasia. Cyclin-dependent kinase 4 (CDK4), a key regulator of G1-phase of the cell cycle, has been shown to be overexpressed in pancreatic cancer. Until now, the contribution of CDK4 to tumor maintenance of pancreatic cancer has not been investigated. In this study, we used the chemical CDK4 inhibitor 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, as well as RNA interference, to investigate the function of CDK4 in pancreatic cancer cells. Both approaches led to a reduction of pancreatic cancer cell proliferation due to G1-phase cell cycle arrest and Rb activation. Furthermore, we observed increased sensitivity of G1-arrested pancreatic cancer cells towards TRAIL-induced apoptosis. Sensitization towards TRAIL was due to the transcriptional downregulation of survivin. These findings show that a combined sensitizer/inducer strategy may be a potential therapeutic strategy for pancreatic ductal adenocarcinoma.

Inhibitory effect of a matrix metalloproteinase inhibitor on growth and spread of human pancreatic ductal adenocarcinoma evaluated in an orthotopic severe combined immunodeficient (SCID) mouse model

The present study was aimed at evaluating the effect of the matrix metalloproteinase (MMP) inhibitor prinomastat (AG3340) on tumor progression using an orthotopic pancreatic carcinoma model in severe combined immunodeficient mice. In controls, receiving vehicle only, the poorly differentiated ductal adenocarcinoma invaded into adjacent organs and metastasized to different sites in the abdomen and to the lungs. Treatment with prinomastat, intraperitoneally twice daily for 21 days, reduced primary tumor volume significantly to 19.0 (±7.7)% of control, with induction of necrosis, differentiation, and fibrotic tissue in the pancreatic tumors. Invasion was not observed in 63% of prinomastat-treated mice, and metastases were reduced markedly. Surprisingly, prinomastat-treated tumors had on average higher microvessel densities as a consequence of an increased angiogenesis in perinecrotic tumor areas. We conclude that prinomastat is highly effective in inhibiting pancreatic carcinoma growth and progression in an orthotopic cancer model. This model appears to be a valuable tool to investigate the potency of novel antimetastatic strategies in pancreatic ductal adenocarcinoma by specifically targeting certain MMPs.