Aarthi Gopinathan

Inhibition of Hedgehog Signaling Enhances Delivery of Chemotherapy in a Mouse Model of Pancreatic Cancer

Its All in the Delivery Pancreatic cancer is almost universally associated with a poor prognosis, in part because the tumors are resistant to chemotherapeutic drugs. Working with a mouse tumor model that displays many features of the human disease, Olive et al. (p. 1457, published online 21 May; see the Perspective by Olson and Hanahan) found that the tumors were poorly vascularized, a factor likely to impede drug delivery. Treatment of the mice with the chemotherapeutic drug gemcitabine in combination with a drug that depletes tumor-associated stromal tissue led to an increase in tumor vasculature, enhanced delivery of gemcitabine, and a delay in disease progression. Thus, drugs targeting the tumor stroma may merit investigation as a way to enhance the efficacy of conventional chemotherapy for pancreatic cancer. Pancreatic tumors are unresponsive to chemotherapy because their limited vasculature precludes efficient drug delivery. Pancreatic ductal adenocarcinoma (PDA) is among the most lethal human cancers in part because it is insensitive to many chemotherapeutic drugs. Studying a mouse model of PDA that is refractory to the clinically used drug gemcitabine, we found that the tumors in this model were poorly perfused and poorly vascularized, properties that are shared with human PDA. We tested whether the delivery and efficacy of gemcitabine in the mice could be improved by coadministration of IPI-926, a drug that depletes tumor-associated stromal tissue by inhibition of the Hedgehog cellular signaling pathway. The combination therapy produced a transient increase in intratumoral vascular density and intratumoral concentration of gemcitabine, leading to transient stabilization of disease. Thus, inefficient drug delivery may be an important contributor to chemoresistance in pancreatic cancer.

Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis

Reactive oxygen species (ROS) are mutagenic and may thereby promote cancer. Normally, ROS levels are tightly controlled by an inducible antioxidant program that responds to cellular stressors and is predominantly regulated by the transcription factor Nrf2 (also known as Nfe2l2) and its repressor protein Keap1 (refs 2–5). In contrast to the acute physiological regulation of Nrf2, in neoplasia there is evidence for increased basal activation of Nrf2. Indeed, somatic mutations that disrupt the Nrf2–Keap1 interaction to stabilize Nrf2 and increase the constitutive transcription of Nrf2 target genes were recently identified, indicating that enhanced ROS detoxification and additional Nrf2 functions may in fact be pro-tumorigenic. Here, we investigated ROS metabolism in primary murine cells following the expression of endogenous oncogenic alleles of Kras, Braf and Myc, and found that ROS are actively suppressed by these oncogenes. K-RasG12D, B-RafV619E and MycERT2 each increased the transcription of Nrf2 to stably elevate the basal Nrf2 antioxidant program and thereby lower intracellular ROS and confer a more reduced intracellular environment. Oncogene-directed increased expression of Nrf2 is a new mechanism for the activation of the Nrf2 antioxidant program, and is evident in primary cells and tissues of mice expressing K-RasG12D and B-RafV619E, and in human pancreatic cancer. Furthermore, genetic targeting of the Nrf2 pathway impairs K-RasG12D-induced proliferation and tumorigenesis in vivo. Thus, the Nrf2 antioxidant and cellular detoxification program represents a previously unappreciated mediator of oncogenesis.

Suppression of Antitumor Immunity by Stromal Cells Expressing Fibroblast Activation Protein–α

Tumor Vaccination Success Vaccination with tumor-specific antigens is one of several attempted therapies seeking to harness the immune system, but—unfortunately—this strategy has been unsuccessful, possibly because of the immunosuppressive properties of the tumor microenvironment. Kraman et al. (p. 827; see the Perspective by Schreiber and Rowley) have identified immunosuppressive cells of mesenchymal origin in mice comprising 2% of the tumor stromal cell population. They were identified by expression of the fibroblast activation protein–α. Deletion of these cells in lung or pancreatic cancers in mice allowed successful therapeutic vaccination against the tumors, which was dependent on the adaptive immune system and the cytokines interferon-γ and tumor necrosis factor–α. These findings reveal that multiple cell types contribute to the immunosuppressive tumor microenvironment and will inform therapeutic cancer vaccine design. Tumor connective-tissue cells of mesenchymal origin suppress antitumor immune responses. The stromal microenvironment of tumors, which is a mixture of hematopoietic and mesenchymal cells, suppresses immune control of tumor growth. A stromal cell type that was first identified in human cancers expresses fibroblast activation protein–α (FAP). We created a transgenic mouse in which FAP-expressing cells can be ablated. Depletion of FAP-expressing cells, which made up only 2% of all tumor cells in established Lewis lung carcinomas, caused rapid hypoxic necrosis of both cancer and stromal cells in immunogenic tumors by a process involving interferon-γ and tumor necrosis factor–α. Depleting FAP-expressing cells in a subcutaneous model of pancreatic ductal adenocarcinoma also permitted immunological control of growth. Therefore, FAP-expressing cells are a nonredundant, immune-suppressive component of the tumor microenvironment.

The pancreas cancer microenvironment

Pancreatic ductal adenocarcinoma (PDA) is a common and lethal malignancy resulting in more than 250,000 deaths per year worldwide. Despite extensive efforts, cytotoxic and targeted therapies have provided only limited efficacy for patients with PDA to date. One contributing factor to the failure of systemic therapies may be the abundant tumor stromal content that is the characteristic of PDA. The PDA stroma, aptly termed the tumor microenvironment, occupies the majority of the tumor mass, and consists of a dynamic assortment of extracellular matrix components and nonneoplastic cells including fibroblastic, vascular, and immune cells. Recent work has revealed that the PDA stroma supports tumor growth and promotes metastasis and simultaneously serves as a physical barrier to drug delivery. Accordingly, methods that alter stromal composition or function, for instance interference with the vasculature via Notch/Hedgehog pathway inhibition or relief of vascular compression by hyaluronidase, are under active investigation. Here, we will review our current understanding of the PDA tumor microenvironment, and highlight opportunities for further exploration that may benefit patients. Clin Cancer Res; 18(16); 4266–76. ©2012 AACR.

Mist1-KrasG12D Knock-In Mice Develop Mixed Differentiation Metastatic Exocrine Pancreatic Carcinoma and Hepatocellular Carcinoma

Despite the prevalence of oncogenic Kras mutations in the earliest stages of pancreatic ductal adenocarcinoma, the cellular compartment in which oncogenic Kras initiates tumorigenesis remains unknown. To address this, we have gene targeted KrasG12D into the open reading frame of Mist1, a basic helix-loop-helix transcription factor that is expressed during pancreatic development and required for proper pancreatic acinar organization. Although the pancreata of Mist1(KrasG12D/+) mutant mice predictably exhibited acinar metaplasia and dysplasia, the frequent death of these mice from invasive and metastatic pancreatic cancer with mixed histologic characteristics, including acinar, cystic, and ductal features, was unexpected and in contrast to previously described mutant mice that ectopically expressed the Kras oncogene in either acinar or ductal compartments. Interestingly, many of the mutant mice developed hepatocellular carcinoma, implicating Mist1(KrasG12D/+) cells in both pancreatic and hepatic neoplasia. Concomitant Trp53+/- mutation cooperated with Mist1(KrasG12D/+) to accelerate lethality and was associated with advanced histopathologic findings, including parenchymal liver metastasis. These findings suggest that Mist1-expressing cells represent a permissive compartment for transformation by oncogenic Kras in pancreatic tumorigenesis.

Gamma secretase inhibition promotes hypoxic necrosis in mouse pancreatic ductal adenocarcinoma

Blocking Notch signaling in pancreatic cancer promotes hypoxia and cell death.

Cathepsin B promotes the progression of pancreatic ductal adenocarcinoma in mice

Objective The lysosomal protease cathepsin B is upregulated in human pancreatic ductal adenocarcinoma (PDA) and represents a potential therapeutic target. Loss of cathepsin B delays tumour progression in mouse models of islet, mammary and intestinal carcinoma and decreases invasion and metastasis. This study examines the role of cathepsin B in the initiation, progression and metastasis of PDA. Methods Cathepsin B germline knockout mice were crossed with animals expressing an endogenous KrasG12D allele in the pancreas, and mice were aged to evaluate the role of cathepsin B in pancreatic intraepithelial neoplasia (PanIN). A survival study was also performed with mice carrying an additional heterozygous conditional Trp53R172H allele. Cell lines derived from tumours were used to investigate the role of cathepsin B in vitro, and subcutaneous allografts investigated the cell autonomous and non-cell autonomous roles of cathepsin B in pancreatic cancer. Results Constitutive cathepsin B loss resulted in delayed progression of both PanIN and PDA and a significant survival advantage in mice. Cathepsin B-deficient PDA cells and PanIN showed decreased proliferation and mitogen-activated protein (MAP) kinase signalling. The reconstitution of deficient cells with cathepsin B reversed these findings, which correlated with decreased levels of the active forms of the related protease cathepsin L. Conversely, acute ablation of cathepsin L activated the MAP kinase cascade in PDA cells. Conclusions These results confirm that cathepsin B plays an important cell autonomous role in the progression of PDA and suggest that the regulation of cathepsin L by cathepsin B may be a means of stimulating cell proliferation in neoplasia.

MRI with hyperpolarised [1-13C]pyruvate detects advanced pancreatic preneoplasia prior to invasive disease in a mouse model

Objectives Pancreatic cancer (PCa) is treatable by surgery when detected at an early stage. Non-invasive imaging methods able to detect both established tumours and their precursor lesions are needed to select patients for surgery. We investigated here whether pancreatic preneoplasia could be detected prior to the development of invasive cancers in genetically engineered mouse models of PCa using metabolic imaging. Design The concentrations of alanine and lactate and the activities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) were measured in extracts prepared from the pancreas of animals at different stages of disease progression; from pancreatitis, through tissue with predominantly low-grade and then high-grade pancreatic intraepithelial neoplasia and then tumour. 13C magnetic resonance spectroscopic imaging (13C-MRSI) was used to measure non-invasively changes in 13C labelling of alanine and lactate with disease progression, following injection of hyperpolarised [1-13C]pyruvate. Results Progressive decreases in the alanine/lactate concentration ratio and ALT/LDH activity ratio with disease progression were accompanied by a corresponding decrease in the [1-13C]alanine/[1-13C]lactate signal ratio observed in 13C-MRSI images of the pancreas. Conclusions Metabolic imaging with hyperpolarised [1-13C]pyruvate enables detection and monitoring of the progression of PCa precursor lesions. Translation of this MRI technique to the clinic has the potential to improve the management of patients at high risk of developing PCa.

GEMMs as preclinical models for testing pancreatic cancer therapies

ABSTRACT Pancreatic ductal adenocarcinoma is the most common form of pancreatic tumour, with a very limited survival rate and currently no available disease-modifying treatments. Despite recent advances in the production of genetically engineered mouse models (GEMMs), the development of new therapies for pancreatic cancer is still hampered by a lack of reliable and predictive preclinical animal models for this disease. Preclinical models are vitally important for assessing therapies in the first stages of the drug development pipeline, prior to their transition to the clinical arena. GEMMs carry mutations in genes that are associated with specific human diseases and they can thus accurately mimic the genetic, phenotypic and physiological aspects of human pathologies. Here, we discuss different GEMMs of human pancreatic cancer, with a focus on the Lox-Stop-Lox (LSL)-KrasG12D; LSL-Trp53R172H; Pdx1-cre (KPC) model, one of the most widely used preclinical models for this disease. We describe its application in preclinical research, highlighting its advantages and disadvantages, its potential for predicting clinical outcomes in humans and the factors that can affect such outcomes, and, finally, future developments that could advance the discovery of new therapies for pancreatic cancer. Drug Discovery Collection: This Review discusses GEMMs of human pancreatic cancer, particularly focusing on the KPC model, its use in preclinical research, advantages and disadvantages, and its potential for predicting clinical outcomes.

Fibroblast drug scavenging increases intratumoural gemcitabine accumulation in murine pancreas cancer

Objective Desmoplasia and hypovascularity are thought to impede drug delivery in pancreatic ductal adenocarcinoma (PDAC). However, stromal depletion approaches have failed to show clinical responses in patients. Here, we aimed to revisit the role of the tumour microenvironment as a physical barrier for gemcitabine delivery. Design Gemcitabine metabolites were analysed in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) murine tumours and matched liver metastases, primary tumour cell lines, cancer-associated fibroblasts (CAFs) and pancreatic stellate cells (PSCs) by liquid chromatography-mass spectrometry/mass spectrometry. Functional and preclinical experiments, as well as expression analysis of stromal markers and gemcitabine metabolism pathways were performed in murine and human specimen to investigate the preclinical implications and the mechanism of gemcitabine accumulation. Results Gemcitabine accumulation was significantly enhanced in fibroblast-rich tumours compared with liver metastases and normal liver. In vitro, significantly increased concentrations of activated 2′,2′-difluorodeoxycytidine-5′-triphosphate (dFdCTP) and greatly reduced amounts of the inactive gemcitabine metabolite 2′,2′-difluorodeoxyuridine were detected in PSCs and CAFs. Mechanistically, key metabolic enzymes involved in gemcitabine inactivation such as hydrolytic cytosolic 5′-nucleotidases (Nt5c1A, Nt5c3) were expressed at low levels in CAFs in vitro and in vivo, and recombinant expression of Nt5c1A resulted in decreased intracellular dFdCTP concentrations in vitro. Moreover, gemcitabine treatment in KPC mice reduced the number of liver metastases by >50%. Conclusions Our findings suggest that fibroblast drug scavenging may contribute to the clinical failure of gemcitabine in desmoplastic PDAC. Metabolic targeting of CAFs may thus be a promising strategy to enhance the antiproliferative effects of gemcitabine.