Faculty Bibliography

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive tumor stroma that is composed of immune cells, vascular cells, fibroblasts and extracellular matrix components (Erkan et al., 2012). This increased desmoplasia correlates with decreased survival and has been shown to mediate chemoresistance (von Hoff et al., 2009). Macrophages within the stromal compartment have been shown to play tumor-promoting roles by switching from their classical immunostimulatory function ('M1') to an immunosuppressive state ('M2') (Yoshikawa et al., 2012). Nab-Paclitaxel (nab-Ptx) is an albumin-bound form of Paclitaxel that, in combination with Gemcitabine, is currently accepted as the standard chemotherapeutic regimen for pancreatic cancer. However, pre-clinical and clinical studies have suggested that nab-Ptx may exert its effects by altering the tumor stroma (Desai et al., 2006, von Hoff et al., 2011). Here, we show that tumor-associated macrophages uptake high levels of nab-Ptx in an orthotopic model of PDAC via macropinocytosis. Eighty to ninety percent of macrophages within the tumor internalize fluorescently labeled nab-Ptx ex vivo, as compared to thirty to forty percent of macrophages from the spleens of the same animals. These data suggest that M2-like macrophages within the tumor microenvironment preferentially macropinocytose nab-Ptx. To analyze the potential consequence of nab-Ptx internalization on macrophages, we treated the RAW macrophage cell line with Ptx alone or in combination with the immunostimulatory cytokine IFN gamma (IFNgamma). Prolonged exposure (48h) of RAW cells to Ptx induced an increase in the M1 marker iNOS, with the combination of low dose IFNgamma and Ptx resulting in a synergistic increase in iNOS expression with accelerated kinetics (12h). Moreover, Ptx alone or in combination with IFNgamma was able to revert IL-4-induced expression of the M2 marker Arginase 1. These findings suggest that high levels of nab-Ptx internalization by M2 macrophages may re-polarize them to an M1, immunostimulatory state. Our in vitro studies suggest that the potential effect of nab-Ptx and Gemcitabine on macrophage polarization in vivo may be enhanced by the presence of an additional immunostimulatory signal. The agonist CD40 monoclonal antibody (mAb) is a member on the TNFalpha receptor superfamily that has been shown to induce immune cell activation and therapeutic efficacy in human and mouse models of PDAC (Beatty et al., 2013). We therefore examined the effect of combining CD40 mAb with nab-Ptx and Gemcitabine treatment on macrophage phenotype in an orthotopic model of PDAC. Our studies to date show that the induction of both an increase in M1 marker (MHCII and CD86) expression and a decrease in M2 marker expression (CD206) in pancreas tumor-associated macrophages requires the triple combination therapy. Together, these data suggest that the internalization of nab-Ptx by tumor-associated macrophages in combination with immune activating signals like CD40 mAb may be required to restore their M1-like, tumor cell cytotoxic functions. CD40 mAb and nab-Ptx combination therapy may therefore enable the effective targeting of the pancreatic tumor stroma, resulting in enhanced therapeutic benefit in PDAC

Apoptotic extrusion is a multicellular process utilized by live cells to remove neighboring apoptotic cells. In epithelial tissues, this process has been shown to be critical for the preservation of tissue integrity and barrier function [1, 2]. Here we demonstrate that extrusion is driven by the retraction of the apoptotic cell, which, in turn, triggers a transient and coordinated elongation of the neighboring cells. The coordination of cell elongation requires E-cadherin-mediated cell-cell adhesion. Accordingly, cells that express low levels of E-cadherin are compromised in elongation and apoptotic extrusion, and furthermore, display loss of barrier function in response to apoptotic stimuli. These findings indicate that the maintenance of adhesive forces during apoptotic cell turnover might play an essential role in controlling tissue homeostasis.

Mutations in KRAS are prevalent in human cancers and universally predictive of resistance to anticancer therapeutics. Although it is widely accepted that acquisition of an activating mutation endows RAS genes with functional autonomy, recent studies suggest that the wild-type forms of Ras may contribute to mutant Ras-driven tumorigenesis. Here, we show that downregulation of wild-type H-Ras or N-Ras in mutant K-Ras cancer cells leads to hyperactivation of the Erk/p90RSK and PI3K/Akt pathways and, consequently, the phosphorylation of Chk1 at an inhibitory site, Ser 280. The resulting inhibition of ATR/Chk1 signaling abrogates the activation of the G2 DNA damage checkpoint and confers specific sensitization of mutant K-Ras cancer cells to DNA damage chemotherapeutic agents in vitro and in vivo.

Macropinocytosis serves as an internalization pathway for extracellular fluid and its contents. Macropinocytosis is upregulated in oncogene-expressing cells and, recently, we have revealed a functional role for macropinocytosis in fueling cancer cell growth through the internalization of extracellular albumin, which is degraded into a usable source of intracellular amino acids. Assessing macropinocytosis has been challenging in the past because of the lack of reliable assays capable of quantitatively measuring this uptake mechanism. Here we describe a protocol for visualizing and quantifying the extent of macropinocytosis in cells both in culture and growing in vivo as tumor xenografts. By using this approach, the 'macropinocytic index' of a particular cell line or subcutaneous tumor can be ascertained within 1-2 d. The protocol can be carried out with multiple samples in parallel and can be easily adapted for a variety of cell types and xenograft or allograft mouse models.

BACKGROUND: The BRAF V600E (BRAF+) mutation activates the mitogen-activated protein kinase (MAPK/ERK) pathway and may confer an aggressive phenotype in papillary thyroid cancer (PTC). Clinically, the behavior of BRAF+ PTC, however, varies from an indolent to an aggressive course. SPRY2 is a negative feedback regulator of the MAPK/ERK pathway. We hypothesize that the level of SPRY2 expression contributes to MAPK/ERK pathway output and accounts for BRAF+ and clinical heterogeneity. METHODS: A tissue microarray with BRAF-positive PTCs (BRAF+ PTCs) was constructed and analyzed for SPRY2 expression and MAPK/ERK output. Data were studied in the context of clinicopathologic factors to develop a risk stratification system predictive of tumor biology. SPRY2 function was studied by silencing SPRY2 in BRAF+ PTC cells. These cells were treated with MAPK/ERK pathway inhibitors and assessed for growth effects. RESULTS: BRAF+ PTCs with an intact MAPK/ERK feedback pathway do not exhibit lymph node metastases. BRAF+ PTCs with dysregulated feedback pathways have nodal metastasis. When SPRY2 is silenced, the BRAF+ PTC cells are significantly more sensitive to MAPK/ERK inhibition. CONCLUSION: PTC behavior likely is dependent on both the driver of the MAPK/ERK pathway and its regulatory feedback. When the feedback pathway is intact, the tumor phenotype seems to be less aggressive. This observation has direct and important clinical implications and may alter our treatment strategies.

RAS is the most frequently mutated oncogene in human cancers. Despite decades of effort, anti-RAS therapies have remained elusive. Isoprenylcysteine carboxylmethyltransferase (ICMT) methylates RAS and other CaaX-containing proteins, but its potential as a target for cancer therapy has not been fully evaluated. We crossed a Pdx1-Cre;LSL-KrasG12D mouse, which is a model of pancreatic ductal adenocarcinoma (PDA), with a mouse harboring a floxed allele of Icmt. Surprisingly, we found that ICMT deficiency dramatically accelerated the development and progression of neoplasia. ICMT-deficient pancreatic ductal epithelial cells had a slight growth advantage and were resistant to premature senescence by a mechanism that involved suppression of cyclin-dependent kinase inhibitor 2A (p16INK4A) expression. ICMT deficiency precisely phenocopied Notch1 deficiency in the Pdx1-Cre;LSL-KrasG12D model by exacerbating pancreatic intraepithelial neoplasias, promoting facial papillomas, and derepressing Wnt signaling. Silencing ICMT in human osteosarcoma cells decreased Notch1 signaling in response to stimulation with cell-surface ligands. Additionally, targeted silencing of Ste14, the Drosophila homolog of Icmt, resulted in defects in wing development, consistent with Notch loss of function. Our data suggest that ICMT behaves like a tumor suppressor in PDA because it is required for Notch1 signaling.