Christine Feig

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.

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.

Hyaluronan impairs vascular function and drug delivery in a mouse model of pancreatic cancer

Objective Pancreatic ductal adenocarcinoma (PDA) is characterised by stromal desmoplasia and vascular dysfunction, which critically impair drug delivery. This study examines the role of an abundant extracellular matrix component, the megadalton glycosaminoglycan hyaluronan (HA), as a novel therapeutic target in PDA. Methods Using a genetically engineered mouse model of PDA, the authors enzymatically depleted HA by a clinically formulated PEGylated human recombinant PH20 hyaluronidase (PEGPH20) and examined tumour perfusion, vascular permeability and drug delivery. The preclinical utility of PEGPH20 in combination with gemcitabine was assessed by short-term and survival studies. Results PEGPH20 rapidly and sustainably depleted HA, inducing the re-expansion of PDA blood vessels and increasing the intratumoral delivery of two chemotherapeutic agents, doxorubicin and gemcitabine. Moreover, PEGPH20 triggered fenestrations and interendothelial junctional gaps in PDA tumour endothelia and promoted a tumour-specific increase in macromolecular permeability. Finally, combination therapy with PEGPH20 and gemcitabine led to inhibition of PDA tumour growth and prolonged survival over gemcitabine monotherapy, suggesting immediate clinical utility. Conclusions The authors demonstrate that HA impedes the intratumoral vasculature in PDA and propose that its enzymatic depletion be explored as a means to improve drug delivery and response in patients with pancreatic cancer.

Stromal biology and therapy in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDA) is an almost uniformly lethal disease. One explanation for the devastating prognosis is the failure of many chemotherapies, including the current standard of care therapy gemcitabine. Although our knowledge of the molecular events underlying multistep carcinogenesis in PDA has steadily increased, translation into more effective therapeutic approaches has been inefficient over the last several decades. Evidence for this innate resistance to systemic therapies was recently provided in an accurate mouse model of PDA by the demonstration that chemotherapies are poorly delivered to PDA tissues because of a deficient vasculature. This vascular deficiency correlated with the presence of a dense stromal matrix that is a prominent histological hallmark of PDA tumours. Therapeutic targeting of stromal cells decreased the stroma from pancreatic tumours, resulting in increased intratumoral perfusion and therapeutic delivery of gemcitabine. Stromal cells contained within the PDA tumour microenvironment therefore represent an additional constituent to neoplastic cells that should be critically evaluated for optimal therapeutic development in preclinical models and early clinical trials.

Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti–PD-L1 immunotherapy in pancreatic cancer

Significance Cancer immune evasion is well described. In some cases, this may be overcome by enhancing T-cell responses. We show that despite the presence of antitumor T cells, immunotherapeutic antibodies are ineffective in a murine pancreatic cancer model recapitulating the human disease. Removing the carcinoma-associated fibroblast (CAF) expressing fibroblast activation protein (FAP) from tumors permitted immune control of tumor growth and uncovered the efficacy of these immunotherapeutic antibodies. FAP+ CAFs are the only tumoral source of chemokine (C-X-C motif) ligand 12 (CXCL12), and administering AMD3100, an inhibitor of chemokine (C-X-C motif) receptor 4, a CXCL12 receptor, also revealed the antitumor effects of an immunotherapeutic antibody and greatly diminished cancer cells. These findings may have wide clinical relevance because FAP+ cells are found in almost all human adenocarcinomas. An autochthonous model of pancreatic ductal adenocarcinoma (PDA) permitted the analysis of why immunotherapy is ineffective in this human disease. Despite finding that PDA-bearing mice had cancer cell-specific CD8+ T cells, the mice, like human patients with PDA, did not respond to two immunological checkpoint antagonists that promote the function of T cells: anti-cytotoxic T-lymphocyte-associated protein 4 (α-CTLA-4) and α-programmed cell death 1 ligand 1 (α-PD-L1). Immune control of PDA growth was achieved, however, by depleting carcinoma-associated fibroblasts (CAFs) that express fibroblast activation protein (FAP). The depletion of the FAP+ stromal cell also uncovered the antitumor effects of α-CTLA-4 and α-PD-L1, indicating that its immune suppressive activity accounts for the failure of these T-cell checkpoint antagonists. Three findings suggested that chemokine (C-X-C motif) ligand 12 (CXCL12) explained the overriding immunosuppression by the FAP+ cell: T cells were absent from regions of the tumor containing cancer cells, cancer cells were coated with the chemokine, CXCL12, and the FAP+ CAF was the principal source of CXCL12 in the tumor. Administering AMD3100, a CXCL12 receptor chemokine (C-X-C motif) receptor 4 inhibitor, induced rapid T-cell accumulation among cancer cells and acted synergistically with α-PD-L1 to greatly diminish cancer cells, which were identified by their loss of heterozygosity of Trp53 gene. The residual tumor was composed only of premalignant epithelial cells and inflammatory cells. Thus, a single protein, CXCL12, from a single stromal cell type, the FAP+ CAF, may direct tumor immune evasion in a model of human PDA.

Let-7 expression defines two differentiation stages of cancer

The early phases of carcinogenesis resemble embryonic development, often involving the reexpression of embryonic mesenchymal genes. The NCI60 panel of human tumor cell lines can genetically be subdivided into two superclusters (SCs) that correspond to CD95 Type I and II cells. SC1 cells are characterized by a mesenchymal and SC2 cells by an epithelial gene signature, suggesting that SC1 cells represent less differentiated, advanced stages of cancer. miRNAs are small 20- to 22-nucleotide-long noncoding RNAs that inhibit gene expression at the posttranscriptional level. By performing miRNA expression analysis on 10 Type I and 10 Type II cells, we have determined that SC1 cells express low and SC2 cells high levels of the miRNA let-7, respectively, suggesting that let-7 is a marker for less advanced cancers. Expression of the let-7 target high-mobility group A2 (HMGA2), an early embryonic gene, but not of classical epithelial or mesenchymal markers such as E-cadherin or vimentin, inversely correlated with let-7 expression in SC1 and SC2 cells. Using ovarian cancer as a model, we demonstrate that expression of let-7 and HMGA2 is a better predictor of prognosis than classical markers such as E-cadherin, vimentin, and Snail. These data identify loss of let-7 expression as a marker for less differentiated cancer.

CD95 promotes tumour growth

CD95 (also called Fas and APO-1) is a prototypical death receptor that regulates tissue homeostasis mainly in the immune system through the induction of apoptosis. During cancer progression CD95 is frequently downregulated or cells are rendered apoptosis resistant, raising the possibility that loss of CD95 is part of a mechanism for tumour evasion. However, complete loss of CD95 is rarely seen in human cancers and many cancer cells express large quantities of CD95 and are highly sensitive to CD95-mediated apoptosis in vitro. Furthermore, cancer patients frequently have elevated levels of the physiological ligand for CD95, CD95L. These data raise the possibility that CD95 could actually promote the growth of tumours through its non-apoptotic activities. Here we show that cancer cells in general, regardless of their CD95 apoptosis sensitivity, depend on constitutive activity of CD95, stimulated by cancer-produced CD95L, for optimal growth. Consistently, loss of CD95 in mouse models of ovarian cancer and liver cancer reduces cancer incidence as well as the size of the tumours. The tumorigenic activity of CD95 is mediated by a pathway involving JNK and Jun. These results demonstrate that CD95 has a growth-promoting role during tumorigenesis and indicate that efforts to inhibit its activity rather than to enhance it should be considered during cancer therapy.

The role of receptor internalization in CD95 signaling

Activation of the cell surface CD95 receptor triggers a cascade of signaling events, including assembly of the death‐inducing signaling complex (DISC), that culminate in cellular apoptosis. In this study, we demonstrate a general requirement of receptor internalization for CD95 ligand‐mediated DISC amplification, caspase activation and apoptosis in type I cells. Recruitment of DISC components to the activated receptor predominantly occurs after the receptor has moved into an endosomal compartment and blockade of CD95 internalization impairs DISC formation and apoptosis. In contrast, CD95 ligand stimulation of cells unable to internalize CD95 results in activation of proliferative Erk and NF‐κB signaling pathways. Hence, the subcellular localization and internalization pathways of CD95 play important roles in controlling activation of distinct signaling cascades to determine divergent cellular fates.

Let-7 prevents early cancer progression by suppressing expression of the embryonic gene HMGA2

The microRNA let-7 regulates late embryonic development by suppressing expression of a number of genes such as c-myc and RAS as well as the embryonic gene high mobility group, A2 (HMGA2). We now demonstrate that HMGA2 is more efficiently targeted by let-7 than RAS. Its expression inversely correlates with the expression of let-7 in the NCI60 cells lines, and the expression of RAS does not change when amounts of let-7 that efficiently silence expression of HMGA2 are introduced into tumor cells. We did not find a difference in the expression of HMGA2 between primary ovarian cancer samples and matching metastases, suggesting that the expression of HMGA2 represents an early event during cancer progression. The late repression of HMGA2 by let-7 during embryonic development, and the early reexpression of HMGA2 during cancer development, is in line with the hypothesis that cancer development represents a case of reverse embryogenesis.

Identification of Let-7–Regulated Oncofetal Genes

MicroRNAs (miRNA) are small RNA molecules of approximately 20 to 22 nucleotides that reduce expression of proteins through mRNA degradation and/or translational silencing. Each known miRNA has a large number of predicted targets. Members of the let-7/miR-98 family of miRNAs are up-regulated at the end of embryonic development. Let-7 is often down-regulated early during cancer development, suggesting that let-7-regulated oncofetal genes (LOG) may become reexpressed in cancer cells. Using comparative bioinformatics, we have identified 12 conserved LOGs that include HMGA2 and IMP-1/CRD-BP. IMP-1 has growth-promoting activities through stabilization of c-myc mRNA. We experimentally confirmed that IMP-1 is a direct let-7 target that promotes cell growth and motility of tumor cells, and we confirmed by proteomics analysis that IMP-1 and HMGA2 are major miRNA targets. Our data suggest that a substantial part of the growth inhibitory activities of let-7 comes from suppressing the expression of IMP-1. LOGs could be novel therapeutic targets and potential biomarkers for cancer treatment.