Jason R. Gunn

Connective tissue growth factor–specific antibody attenuates tumor growth, metastasis, and angiogenesis in an orthotopic mouse model of pancreatic cancer

Connective tissue growth factor (CTGF) plays an important role in fibrosis by modulating cell migration and cell growth but may also modify tumor growth and metastasis. Because CTGF is overexpressed in pancreatic ductal adenocarcinoma, we investigated the in vitro effects of CTGF on the proliferation and invasiveness of PANC-1 pancreatic cancer cells and examined the consequences of its in vivo inhibition on the growth and metastasis of these cells using a fully human CTGF-specific monoclonal antibody (FG-3019) in an orthotopic nude mouse model. Although PANC-1 cells expressed relatively high levels of endogenous CTGF mRNA, the addition of CTGF to conditioned medium increased the proliferation and invasiveness of PANC-1 cells. Moreover, transforming growth factor-β1 caused a further increase in CTGF expression in these cells. In vivo, the twice weekly i.p. administration of FG-3019 decreased tumor growth and metastasis and attenuated tumor angiogenesis and cancer cell proliferation. FG-3019 did not enhance apoptosis and did not attenuate the inhibitory effects of gemcitabine on tumor growth and metastasis. These findings suggest that CTGF may contribute to aberrant autocrine and paracrine pathways that promote pancreatic cancer cell growth, invasion, metastasis, and angiogenesis. Therefore, blocking CTGF actions with FG-3019 may represent a novel therapeutic approach in pancreatic ductal adenocarcinoma. [Mol Cancer Ther 2006;5(5):1108–16]

Acute pancreatitis markedly accelerates pancreatic cancer progression in mice expressing oncogenic Kras

Chronic pancreatitis increases by 16-fold the risk of developing pancreatic ductal adenocarcinoma (PDAC), one of the deadliest human cancers. It also appears to accelerate cancer progression in genetically engineered mouse models. We now report that in a mouse model where oncogenic Kras is activated in all pancreatic cell types, two brief episodes of acute pancreatitis caused rapid PanIN progression and accelerated pancreatic cancer development. Thus, a brief inflammatory insult to the pancreas, when occurring in the context of oncogenic Kras(G12D), can initiate a cascade of events that dramatically enhances the risk for pancreatic malignant transformation.

The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces growth inhibition and enhances gemcitabine-induced cell death in pancreatic cancer

Purpose: Pancreatic cancer is an aggressive human malignancy that is generally refractory to chemotherapy. Histone deacetylase inhibitors are novel agents that modulate cell growth and survival. In this study, we sought to determine whether a relatively new histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), inhibits pancreatic cancer cell growth. Experimental Design: The effects of SAHA on the growth of three pancreatic cancer cell lines (BxPC3, COLO-357, and PANC-1) were examined with respect to cell cycle progression, p21 induction and localization, and interactions with the nucleoside analogue gemcitabine. Results: SAHA induced a G1 cell cycle arrest in BxPC-3 cells and COLO-357 cells but not in PANC-1 cells. This arrest was dependent, in part, on induction of p21 by SAHA, as p21 was not induced in PANC-1 cells, and knockdown of p21 using small interfering RNA oligonucleotides nearly completely suppressed the effects of SAHA on cell cycle arrest in COLO-357 and partly attenuated the effects of SAHA in BxPC-3. COLO-357 and BxPC-3 cells, but not PANC-1 cells, were also sensitive to gemcitabine. In the gemcitabine-resistant PANC-1 cells, a 48-h cotreatment with SAHA rendered the cells sensitive to the inhibitory and proapoptotic effects of gemcitabine. An additive effect on growth inhibition by SAHA and gemcitabine was observed in COLO-357 and BxPC-3 cells. Moreover, analysis of p21 distribution in COLO-357 cells revealed that SAHA induced the cytoplasmic localization of both p21 and phospho-p21. Conclusions: These data indicate that SAHA exerts proapoptotic effects in pancreatic cancer cells, in part, by up-regulating p21 and sequestering it in the cytoplasm, raising the possibility that SAHA may have therapeutic potential in the treatment of pancreatic cancer.

Glypican-1 modulates the angiogenic and metastatic potential of human and mouse cancer cells

Cells isolated from many types of human cancers express heparin-binding growth factors (HBGFs) that drive tumor growth, metastasis, and angiogenesis. The heparan sulfate proteoglycan glypican-1 (GPC1) is a coreceptor for HBGFs. Here we show that both cancer cell-derived and host-derived GPC1 are crucial for efficient growth, metastasis, and angiogenesis of human and mouse cancer cells. Thus downregulation of GPC1 in the human pancreatic cancer cell line PANC-1, using antisense approaches, resulted in prolonged doubling times and decreased anchorage-independent growth in vitro as well as attenuated tumor growth, angiogenesis, and metastasis when these cells were transplanted into athymic mice. Moreover, athymic mice that lacked GPC1 exhibited decreased tumor angiogenesis and metastasis following intrapancreatic implantation with either PANC-1 or T3M4 human pancreatic cancer cells and fewer pulmonary metastases following intravenous injection of murine B16-F10 melanoma cells. In addition, hepatic endothelial cells isolated from these mice exhibited an attenuated mitogenic response to VEGF-A. These data indicate that cancer cell- and host-derived GPC1 are crucial for full mitogenic, angiogenic, and metastatic potential of cancer cells. Thus targeting GPC1 might provide new avenues for cancer therapy and for the prevention of cancer metastasis.

In Vivo Quantification of Tumor Receptor Binding Potential with Dual-Reporter Molecular Imaging

PurposeReceptor availability represents a key component of current cancer management. However, no approaches have been adopted to do this clinically, and the current standard of care is invasive tissue biopsy. A dual-reporter methodology capable of quantifying available receptor binding potential of tumors in vivo within a clinically relevant time scale is presented.ProceduresTo test the methodology, a fluorescence imaging-based adaptation was validated against ex vivo and in vitro measures of epidermal growth factor receptor (EGFR) binding potential in four tumor lines in mice, each line expected to express a different level of EGFR.ResultsA strong correlation was observed between in vivo and ex vivo measures of binding potential for all tumor lines (r = 0.99, p < 0.01, slope = 1.80 ± 0.48, and intercept = −0.58 ± 0.84) and between in vivo and in vitro for the three lines expressing the least amount of EGFR (r = 0.99, p < 0.01, slope = 0.64 ± 0.32, and intercept = 0.47 ± 0.51).ConclusionsBy providing a fast and robust measure of receptor density in tumors, the presented methodology has powerful implications for improving choices in cancer intervention, evaluation, and monitoring, and can be scaled to the clinic with an imaging modality like SPECT.

Dynamic dual-tracer MRI-guided fluorescence tomography to quantify receptor density in vivo

The up-regulation of cell surface receptors has become a central focus in personalized cancer treatment; however, because of the complex nature of contrast agent pharmacokinetics in tumor tissue, methods to quantify receptor binding in vivo remain elusive. Here, we present a dual-tracer optical technique for noninvasive estimation of specific receptor binding in cancer. A multispectral MRI-coupled fluorescence molecular tomography system was used to image the uptake kinetics of two fluorescent tracers injected simultaneously, one tracer targeted to the receptor of interest and the other tracer a nontargeted reference. These dynamic tracer data were then fit to a dual-tracer compartmental model to estimate the density of receptors available for binding in the tissue. Applying this approach to mice with deep-seated gliomas that overexpress the EGF receptor produced an estimate of available receptor density of 2.3 ± 0.5 nM (n = 5), consistent with values estimated in comparative invasive imaging and ex vivo studies.

Deletion of Rb accelerates pancreatic carcinogenesis by oncogenic Kras and impairs senescence in premalignant lesions.

BACKGROUND & AIMS Rb1 encodes a cell-cycle regulator that is functionally disrupted in most human cancers. Pancreatic ductal adenocarcinomas (PDACs) have a high frequency of mutations in KRAS and INK4A/CDKN2A that might allow cells to bypass the regulatory actions of retinoblastoma (RB). To determine the role of loss of RB function in PDAC progression, we investigated the effects of Rb disruption during pancreatic malignant transformation initiated by oncogenic Kras. METHODS We generated mice with pancreas-specific disruption of Rb, in the absence or presence of oncogenic Kras, to examine the role of RB in pancreatic carcinogenesis. RESULTS In the presence of oncogenic Kras, loss of Rb from the pancreatic epithelium accelerated formation of pancreatic intraepithelial neoplasia (PanIN), increased the frequency of cystic neoplasms, and promoted rapid progression toward PDAC. Early stage cancers were characterized by acute pancreatic inflammation, associated with up-regulation of proinflammatory cytokines within the pancreas. Despite the presence of markers associated with oncogene-induced senescence, low-grade PanIN were highly proliferative and expressed high levels of p53. Pancreatic cancer cell lines derived from these mice expressed high levels of cytokines, and transcriptional activity of p53 was impaired. CONCLUSIONS Rb encodes a tumor suppressor that attenuates progression of oncogenic Kras-induced carcinogenesis in the pancreas by mediating the senescence response and promoting activity of the tumor suppressor p53.

Microscopic lymph node tumor burden quantified by macroscopic dual-tracer molecular imaging

Lymph node biopsy is employed in many cancer surgeries to identify metastatic disease and to determine cancer stage, yet morbidity and diagnostic delays associated with lymph node biopsy could be avoided if noninvasive imaging of nodal involvement were reliable. Molecular imaging has potential in this regard; however, variable delivery and nonspecific uptake of imaging tracers have made conventional approaches ineffective clinically. Here we present a method of correcting for nonspecific uptake with injection of a second untargeted tracer that allows for quantification of tumor burden in lymph nodes. We confirmed the approach in an athymic mouse model of metastatic human breast cancer by targeting epidermal growth factor receptor, a cell surface receptor overexpressed by many cancers. We observed a significant correlation between in vivo (dual-tracer) and ex vivo measures of tumor burden (r = 0.97, P < 0.01), with an ultimate sensitivity of approximately 200 cells (potentially more sensitive than conventional lymph node biopsy).

Acute Pancreatitis Accelerates Initiation and Progression to Pancreatic Cancer in Mice Expressing Oncogenic Kras in the Nestin Cell Lineage

Targeting of oncogenic Kras to the pancreatic Nestin-expressing embryonic progenitor cells and subsequently to the adult acinar compartment and Nestin-expressing cells is sufficient for the development of low grade pancreatic intraepithelial neoplasia (PanIN) between 2 and 4 months. The mice die around 6 month-old of unrelated causes, and it is therefore not possible to assess whether the lesions will progress to carcinoma. We now report that two brief episodes of caerulein-induced acute pancreatitis in 2 month-old mice causes rapid PanIN progression and pancreatic ductal adenocarcinoma (PDAC) development by 4 months of age. These events occur with similar frequency as observed in animals where the oncogene is targeted during embryogenesis to all pancreatic cell types. Thus, these data show that oncogenic Kras-driven PanIN originating in a non-ductal compartment can rapidly progress to PDAC when subjected to a brief inflammatory insult.

Activated K-ras and INK4a/Arf Deficiency Cooperate During the Development of Pancreatic Cancer by Activation of Notch and NF-κB Signaling Pathways

Background Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, suggesting that novel strategies for the prevention and treatment of PDAC are urgently needed. K-ras mutations are observed in >90% of pancreatic cancer, suggesting its role in the initiation and early developmental stages of PDAC. In order to gain mechanistic insight as to the role of mutated K-ras, several mouse models have been developed by targeting a conditionally mutated K-rasG12D for recapitulating PDAC. A significant co-operativity has been shown in tumor development and metastasis in a compound mouse model with activated K-ras and Ink4a/Arf deficiency. However, the molecular mechanism(s) by which K-ras and Ink4a/Arf deficiency contribute to PDAC has not been fully elucidated. Methodology/Principal Findings To assess the molecular mechanism(s) that are involved in the development of PDAC in the compound transgenic mice with activated K-ras and Ink4a/Arf deficiency, we used multiple methods, such as Real-time RT-PCR, western blotting assay, immunohistochemistry, MTT assay, invasion, EMSA and ELISA. We found that the deletion of Ink4a/Arf in K-rasG12D expressing mice leads to PDAC, which is in part mediated through the activation of Notch and NF-κB signaling pathways. Moreover, we found down-regulation of miR-200 family, which could also play important roles in tumor development and progression of PDAC in the compound transgenic mice. Conclusions/Significance Our results suggest that the activation of Notch and NF-κB together with the loss of miR-200 family is mechanistically linked with the development and progression of PDAC in the compound K-rasG12D and Ink4a/Arf deficient transgenic mice.