Zobeida Cruz-Monserrate

An NF-κB pathway-mediated positive feedback loop amplifies Ras activity to pathological levels in mice

Genetic mutations that give rise to active mutant forms of Ras are oncogenic and found in several types of tumor. However, such mutations are not clear biomarkers for disease, since they are frequently detected in healthy individuals. Instead, it has become clear that elevated levels of Ras activity are critical for Ras-induced tumorigenesis. However, the mechanisms underlying the production of pathological levels of Ras activity are unclear. Here, we show that in the presence of oncogenic Ras, inflammatory stimuli initiate a positive feedback loop involving NF-κB that further amplifies Ras activity to pathological levels. Stimulation of Ras signaling by typical inflammatory stimuli was transient and had no long-term sequelae in wild-type mice. In contrast, these stimuli generated prolonged Ras signaling and led to chronic inflammation and precancerous pancreatic lesions (PanINs) in mice expressing physiological levels of oncogenic K-Ras. These effects of inflammatory stimuli were disrupted by deletion of inhibitor of NF-κB kinase 2 (IKK2) or inhibition of Cox-2. Likewise, expression of active IKK2 or Cox-2 or treatment with LPS generated chronic inflammation and PanINs only in mice expressing oncogenic K-Ras. The data support the hypothesis that in the presence of oncogenic Ras, inflammatory stimuli trigger an NF-κB-mediated positive feedback mechanism involving Cox-2 that amplifies Ras activity to pathological levels. Because a large proportion of the adult human population possesses Ras mutations in tissues including colon, pancreas, and lung, disruption of this positive feedback loop may be an important strategy for cancer prevention.

A high-fat diet activates oncogenic Kras and COX2 to induce development of pancreatic ductal adenocarcinoma in mice

BACKGROUND & AIMS Obesity is a risk factor for pancreatic ductal adenocarcinoma (PDAC), but it is not clear how obesity contributes to pancreatic carcinogenesis. The oncogenic form of KRAS is expressed during early stages of PDAC development and is detected in almost all of these tumors. However, there is evidence that mutant KRAS requires an additional stimulus to activate its full oncogenic activity and that this stimulus involves the inflammatory response. We investigated whether the inflammation induced by a high-fat diet, and the accompanying up-regulation of cyclooxygenase-2 (COX2), increases Kras activity during pancreatic carcinogenesis in mice. METHODS We studied mice with acinar cell-specific expression of KrasG12D (LSL-Kras/Ela-CreERT mice) alone or crossed with COX2 conditional knockout mice (COXKO/LSL-Kras/Ela-CreERT). We also studied LSL-Kras/PDX1-Cre mice. All mice were fed isocaloric diets with different amounts of fat, and a COX2 inhibitor was administered to some LSL-Kras/Ela-CreERT mice. Pancreata were collected from mice and analyzed for Kras activity, levels of phosphorylated extracellular-regulated kinase, inflammation, fibrosis, pancreatic intraepithelial neoplasia (PanIN), and PDACs. RESULTS Pancreatic tissues from LSL-Kras/Ela-CreERT mice fed high-fat diets (HFDs) had increased Kras activity, fibrotic stroma, and numbers of PanINs and PDACs than LSL-Kras/Ela-CreERT mice fed control diets; the mice fed the HFDs also had shorter survival times than mice fed control diets. Administration of a COX2 inhibitor to LSL-Kras/Ela-CreERT mice prevented these effects of HFDs. We also observed a significant reduction in survival times of mice fed HFDs. COXKO/LSL-Kras/Ela-CreERT mice fed HFDs had no evidence for increased numbers of PanIN lesions, inflammation, or fibrosis, as opposed to the increases observed in LSL-Kras/Ela-CreERT mice fed HFDs. CONCLUSIONS In mice, an HFD can activate oncogenic Kras via COX2, leading to pancreatic inflammation and fibrosis and development of PanINs and PDAC. This mechanism might be involved in the association between risk for PDAC and HFDs.

Cell Surface Lactate Receptor GPR81 Is Crucial for Cancer Cell Survival

The mechanisms that allow cancer cells to adapt to the typical tumor microenvironment of low oxygen and glucose and high lactate are not well understood. GPR81 is a lactate receptor recently identified in adipose and muscle cells that has not been investigated in cancer. In the current study, we examined GPR81 expression and function in cancer cells. We found that GPR81 was present in colon, breast, lung, hepatocellular, salivary gland, cervical, and pancreatic carcinoma cell lines. Examination of tumors resected from patients with pancreatic cancer indicated that 94% (148 of 158) expressed high levels of GPR81. Functionally, we observed that the reduction of GPR81 levels using shRNA-mediated silencing had little effect on pancreatic cancer cells cultured in high glucose, but led to the rapid death of cancer cells cultured in conditions of low glucose supplemented with lactate. We also observed that lactate addition to culture media induced the expression of genes involved in lactate metabolism, including monocarboxylase transporters in control, but not in GPR81-silenced cells. In vivo, GPR81 expression levels correlated with the rate of pancreatic cancer tumor growth and metastasis. Cells in which GPR81 was silenced showed a dramatic decrease in growth and metastasis. Implantation of cancer cells in vivo was also observed to lead to greatly elevated levels of GPR81. These data support that GPR81 is important for cancer cell regulation of lactate transport mechanisms. Furthermore, lactate transport is important for the survival of cancer cells in the tumor microenvironment. Cancer Res; 74(18); 5301-10. ©2014 AACR.

Detection of pancreatic cancer tumours and precursor lesions by cathepsin E activity in mouse models

Background and Aims Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the USA. Surgical resection is the only effective treatment; however, only 20% of patients are candidates for surgery. The ability to detect early PDAC would increase the availability of surgery and improve patient survival. This study assessed the feasibility of using the enzymatic activity of cathepsin E (Cath E), a protease highly and specifically expressed in PDAC, as a novel biomarker for the detection of pancreas-bearing pancreatic intraepithelial neoplasia (PanIN) lesions and PDAC. Methods Pancreas from normal, chronic pancreatitis and PDAC patients was assessed for Cath E expression by quantitative real-time PCR and immunohistochemistry. Human PDAC xenografts and genetically engineered mouse models (GEMM) of PDAC were injected with a Cath E activity selective fluorescent probe and imaged using an optical imaging system. Results The specificity of Cath E expression in PDAC patients and GEMM of pancreatic cancer was confirmed by quantitative real-time PCR and immunohistochemistry. The novel probe for Cath E activity specifically detected PDAC in both human xenografts and GEMM in vivo. The Cath E sensitive probe was also able to detect pancreas with PanIN lesions in GEMM before tumour formation. Conclusions The elevated Cath E expression in PanIN and pancreatic tumours allowed in-vivo detection of human PDAC xenografts and imaging of pancreas with PanIN and PDAC tumours in GEMM. Our results support the usefulness of Cath E activity as a potential molecular target for PDAC and early detection imaging.

Targeting Pancreatic Ductal Adenocarcinoma Acidic Microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the USA, accounting for ~40,000 deaths annually. The dismal prognosis for PDAC is largely due to its late diagnosis. Currently, the most sensitive diagnosis of PDAC requires invasive procedures, such as endoscopic ultrasonography, which has inherent risks and accuracy that is highly operator dependent. Here we took advantage of a general characteristic of solid tumors, the acidic microenvironment that is generated as a by-product of metabolism, to develop a novel approach of using pH (Low) Insertion Peptides (pHLIPs) for imaging of PDAC. We show that fluorescently labeled pHLIPs can localize and specifically detect PDAC in human xenografts as well as PDAC and PanIN lesions in genetically engineered mouse models. This novel approach may improve detection, differential diagnosis and staging of PDAC.

Molecular imaging of Cathepsin E-positive tumors in mice using a novel protease-activatable fluorescent probe

UNLABELLED The purpose of this study is to demonstrate the ability of imaging Cathepsin E (Cath E) positive tumors in living animals through selective targeting of Cath E proteolytic activity using a sensitive molecular imaging agent. METHODS A peptide-based Cath E imaging probe and a control probe were synthesized for this study. Human Cath E-positive cancer cells (MPanc96-E) were implanted subcutaneously in nude mice. Tumor-bearing mice were examined in vivo with near-infrared fluorescence (NIRF) imaging at various time points after intravenous injection of the Cath E sensing imaging probe. Excised organs and tissues of interest were further imaged ex vivo. RESULTS Upon specific Cath E proteolytic activation, the NIRF signal of the imaging probe a was converted from an optically quenched initial state to a highly fluorescent active state. Imaging probe a was able to highlight the Cath E-positive tumors as early as 24 h post injection. Fluorescent signal in tumor was 3-fold higher than background. The confined specificity of imaging probe a to tumor associated Cath E was verified by using control imaging probe b. Both in vivo and ex vivo imaging results confirmed the superior selectivity and sensitivity of imaging probe a in Cath E imaging. CONCLUSIONS The small animal studies demonstrated the capability of probe a for imaging Cath E-positive tumors. The developed optical probe could be applied in early diagnostic imaging and guiding subsequent surgical procedure.

Pancreatic cancer-associated Cathepsin E as a drug activator

Pancreatic ductal adenocarcinoma (PDAC) is challenging to treat, and better means to detect and/or treat pancreatic cancer are urgently needed to save lives. Cathepsin E (Cath E) is a proteolytic enzyme highly expressed in PDAC. In this study, a novel approach using Cath E activation of a Cath E-specific prodrug was demonstrated. Specific activation of the prodrug is expected to kill pancreatic cancer cells without harming normal pancreatic cells. A novel 5-aminolevulinic acid (5-ALA) prodrug was custom-designed to be activated selectively by endogenous Cath E within the PDAC cells. The 5-ALA prodrug was incubated with Cath E-positive and -negative tumor cells and illuminated with various doses of light. In addition, mice genetically engineered to develop PDAC were injected intravenously with the 5-ALA prodrug, and the pancreas was treated with light irradiation. One day after treatment, PDAC tissue was assessed for apoptosis. The 5-ALA prodrug was activated within the Cath E-positive tumor but not in the normal pancreatic tissue. When used in combination with light treatment, it allowed delivery of selective photodynamic therapy (PDT) to the cancerous tissues, with minimal harm to the adjacent normal tissues. With this novel Cath E activation approach, it is possible to detect pancreatic cancer cells accurately and specifically impair their viability, while sparing normal cells. This treatment could result in fewer side effects than the non-specific treatments currently in use. Cath E is a specific and effective drug activator for PDAC treatment.

Bisphosphonates Inhibit Stellate Cell Activity and Enhance Antitumor Effects of Nanoparticle Albumin Bound-Paclitaxel in Pancreatic Ductal Adenocarcinoma

Pancreatic stellate cells (PSC) have been recognized as the principal cells responsible for the production of fibrosis in pancreatic ductal adenocarcinoma (PDAC). Recently, PSCs have been noted to share characteristics with cells of monocyte-macrophage lineage (MML cells). Thus, we tested whether PSCs could be targeted with the nitrogen-containing bisphosphonates (NBP; pamidronate or zoledronic acid), which are potent MML cell inhibitors. In addition, we tested NBPs treatment combination with nanoparticle albumin–bound paclitaxel (nab-paclitaxel) to enhance antitumor activity. In vitro, we observed that PSCs possess α-naphthyl butyrate esterase (ANBE) enzyme activity, a specific marker of MML cells. Moreover, NBPs inhibited PSCs proliferation, activation, release of macrophage chemoattractant protein-1 (MCP-1), and type I collagen expression. NBPs also induced PSCs apoptosis and cell-cycle arrest in the G1 phase. In vivo, NBPs inactivated PSCs; reduced fibrosis; inhibited tumor volume, tumor weight, peritoneal dissemination, angiogenesis, and cell proliferation; and increased apoptosis in an orthotopic murine model of PDAC. These in vivo antitumor effects were enhanced when NBPs were combined with nab-paclitaxel but not gemcitabine. Our study suggests that targeting PSCs and tumor cells with NBPs in combination with nab-paclitaxel may be a novel therapeutic approach to PDAC. Mol Cancer Ther; 13(11); 2583–94. ©2014 AACR.

Ductal activation of oncogenic KRAS alone induces sarcomatoid phenotype.

Salivary duct carcinoma (SDC) is an uncommon, but aggressive malignant tumor with a high mortality rate. Herein, we reported the detection of somatic KRAS A146T and Q61H mutations in 2 out of 4 (50%) sarcomatoid SDC variants. Transgenic mice carrying the human oncogenic KRASG12V, which spatiotemporal activation by tamoxifen (TAM)-inducible Cre recombinase Ela-CreERT in the submandibular gland (SMG) ductal cells, was established and characterized. Visible carcinoma was detected as early as day-15 following oncogenic KRASG12V induction alone, and these tumors proliferate rapidly with a median survival of 28-days accompanied with histological reminiscences to human sarcomatoid SDC variants. Moreover, these tumors were resistant to cetuximab treatment despite augmented EGFR signaling, attesting its malignancy. Our findings suggest that LGL-KRasG12V;Ela-CreERT transgenic mice could serve as a useful preclinical model for investigating underlying mechanisms and developing potential therapies.

Abstract B24: Targeting pancreatic ductal adenocarcinoma acidic microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the USA, accounting for ~40,000 deaths annually. The dismal prognosis for PDAC is largely due to its late diagnosis. Currently, the most sensitive diagnosis of PDAC requires invasive procedures, such as endoscopic ultrasonography, which has inherent risks and accuracy that is highly operator dependent. Here we took advantage of a general characteristic of solid tumors, the acidic microenvironment that is generated as a by-product of metabolism, to develop a novel approach of using pH (Low) Insertion Peptides (pHLIPs) for imaging of PDAC. We show that fluorescently labeled pHLIPs can localize and specifically detect PDAC in human xenografts as well as PDAC and PanIN lesions in genetically engineered mouse models. This novel approach may improve detection, differential diagnosis and staging of PDAC Citation Format: Zobeida Cruz-Monserrate, Christina L. Roland, Defeng Deng, Thiruvengadam Arumugam, Anna Moshnikova, Oleg A. Andreev, Yana Reshetnyak, Craig D. Logsdon. Targeting pancreatic ductal adenocarcinoma acidic microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B24.