Mohammed Ghallab

Nuclear translocation of FGFR1 and FGF2 in pancreatic stellate cells facilitates pancreatic cancer cell invasion.

Pancreatic cancer is characterised by desmoplasia, driven by activated pancreatic stellate cells (PSCs). Over‐expression of FGFs and their receptors is a feature of pancreatic cancer and correlates with poor prognosis, but whether their expression impacts on PSCs is unclear. At the invasive front of human pancreatic cancer, FGF2 and FGFR1 localise to the nucleus in activated PSCs but not cancer cells. In vitro, inhibiting FGFR1 and FGF2 in PSCs, using RNAi or chemical inhibition, resulted in significantly reduced cell proliferation, which was not seen in cancer cells. In physiomimetic organotypic co‐cultures, FGFR inhibition prevented PSC as well as cancer cell invasion. FGFR inhibition resulted in cytoplasmic localisation of FGFR1 and FGF2, in contrast to vehicle‐treated conditions where PSCs with nuclear FGFR1 and FGF2 led cancer cells to invade the underlying extra‐cellular matrix. Strikingly, abrogation of nuclear FGFR1 and FGF2 in PSCs abolished cancer cell invasion. These findings suggest a novel therapeutic approach, where preventing nuclear FGF/FGFR mediated proliferation and invasion in PSCs leads to disruption of the tumour microenvironment, preventing pancreatic cancer cell invasion.

Imbalance of desmoplastic stromal cell numbers drives aggressive cancer processes

Epithelial tissues have sparse stroma, in contrast to their corresponding tumours. The effect of cancer cells on stromal cells is well recognized. Increasingly, stromal components, such as endothelial and immune cells, are considered indispensable for cancer progression. The role of desmoplastic stroma, in contrast, is poorly understood. Targeting such cellular components within the tumour is attractive. Recent evidence strongly points towards a dynamic stromal cell participation in cancer progression that impacts patient prognosis. The role of specific desmoplastic stromal cells, such as stellate cells and myofibroblasts in pancreatic, oesophageal and skin cancers, was studied in bio‐engineered, physiomimetic organotypic cultures and by regression analysis. For pancreatic cancer, the maximal effect on increasing cancer cell proliferation and invasion, as well as decreasing cancer cell apoptosis, occurs when stromal (pancreatic stellate cells) cells constitute the majority of the cellular population (maximal effect at a stromal cell proportion of 0.66–0.83), accompanied by change in expression of key molecules such as E‐cadherin and β‐catenin. Gene‐expression microarrays, across three tumour types, indicate that stromal cells consistently and significantly alter global cancer cell functions such as cell cycle, cell–cell signalling, cell movement, cell death and inflammatory response. However, these changes are mediated through cancer type‐specific alteration of expression, with very few common targets across tumour types. As highlighted by these in vitro data, the reciprocal relationship of E‐cadherin and polymeric immunoglobulin receptor (PIGR) expression in cancer cells could be shown, in vivo, to be dependent on the stromal content of human pancreatic cancer. These studies demonstrate that context‐specific cancer–stroma crosstalk requires to be precisely defined for effective therapeutic targeting. These data may be relevant to non‐malignant processes where epithelial cells interact with stromal cells, such as chronic inflammatory and fibrotic conditions. Copyright

Pancreatic cancer organotypics: High throughput, preclinical models for pharmacological agent evaluation.

Pancreatic cancer carries a terrible prognosis, as the fourth most common cause of cancer death in the Western world. There is clearly a need for new therapies to treat this disease. One of the reasons no effective treatment has been developed in the past decade may in part, be explained by the diverse influences exerted by the tumour microenvironment. The tumour stroma cross-talk in pancreatic cancer can influence chemotherapy delivery and response rate. Thus, appropriate preclinical in vitro models which can bridge simple 2D in vitro cell based assays and complex in vivo models are required to understand the biology of pancreatic cancer. Here we discuss the evolution of 3D organotypic models, which recapitulare the morphological and functional features of pancreatic ductal adenocarcinoma (PDAC). Organotypic cultures are a valid high throughput preclinical in vitro model that maybe a useful tool to help establish new therapies for PDAC. A huge advantage of the organotypic model system is that any component of the model can be easily modulated in a short time-frame. This allows new therapies that can target the cancer, the stromal compartment or both to be tested in a model that mirrors the in vivo situation. A major challenge for the future is to expand the cellular composition of the organotypic model to further develop a system that mimics the PDAC environment more precisely. We discuss how this challenge is being met to increase our understanding of this terrible disease and develop novel therapies that can improve the prognosis for patients.

Gastrointestinal stromal tumour presenting with duodenal-jejunal intussusception: a case report

Duodenal-jejunal intussusception is an extremely rare occurrence and has never been reported in the context of a gastrointestinal stromal tumour (GIST). We present the case of a duodenal GIST which presented with major intestinal haemorrhage in addition to duodenal-jejunal intussusception.

Laparoscopic fellowship training can deliver a competent laparoscopic surgeon and trainer.

Aim  The study investigated whether experience gained during a UK laparoscopic colorectal fellowship enabled the fellow subsequently to train consultant colleagues in laparoscopic surgery.

538 Nuclear Translocation of FGFR1 and FGF2 in Pancreatic Stellate Cells Is Necessary for Pancreatic Cancer Cell Invasion

Background: It is now appreciated that, in pancreatic cancer, transformed cells interact with stromal cells, extracellular matrix proteins, and neighboring normal epithelial cells to exploit feedback mechanisms and facilitate tumour progression. Studies have shown that overexpression of FGF2 in pancreatic cancer correlates with poor patient survival and nuclear FGF2 has been observed in pancreatic cancer patient tissue but not in normal patient samples (Yamanka et al 1993). A number of mechanisms have been proposed for the nuclear accumulation of FGFR1 and its ligand; however, the role of intracrine FGF2 signalling in tumour invasion remains poorly understood. We investigated whether nuclear translocation of FGFR1 with FGF2 plays a role in pancreatc stellate cell (PSC) behaviour. Design: FGF2 and FGFR1 RNAi, together with an FGFR inhibitor (PD173074) were used on pancreatic cancer cell lines PSCs and in organotypic cultures to assess their effect on cell behaviour and to assess the effects of blocking FGF signalling on cancer cell behaviour and invasion. Nuclear FGFR1 and FGF2 expression were also studied in vivo. Results: FGF2 and FGFR1 localised to the nucleus in stromal fibroblasts at the invasive front of human pancreatic cancer tissue. In vitro, FGFR1 and FGF2 co-localised to the nucleus in PSCs but not in cancer or normal ductal epithelial cell lines. PSCs but not cancer cells secrete both HMW and LMW FGF2 isoforms. Abolishing nuclear FGFR1 and FGF2 in PSCs using either RNAi or FGFR inhibitor resulted in a significant reduction in cell proliferation which correlated with a G1 cell-cycle block and reduction in the G1 cyclin, cyclin D1. In an organotypic model, nuclear FGFR1 and FGF2 were significantly greater in PSCs invading into the matrix. When treated with FGFR inhibitor, PSCs were unable to invade into the extra-cellular matrix and FGFR1 and FGF2 remained cytoplasmic. Effective blockade of nuclear FGFR1 signalling in PSCs abolished cancer cell invasion. Conclusion: These studies show for the first time that nuclear FGFR1 and FGF2 plays a role in driving pancreatic stellate cell proliferation. Preventing nuclear FGF/FGFR mediated proliferation in PSCs leads to disruption of the tumour microenvironment, thus preventing pancreatic cancer cell invasion.

Abstract B80: Targeting pancreatic cancer stroma with histone deacetylase inhibitors.

Background: Histone deacetylase inhibitors (HDACi) have been shown as a potential therapeutic agent for pancreatic cancer in vitro and in vivo (Donadelli et al. 2003; Fritsche et al. 2009; von Burstin et al. 2009; Schuler et al. 2010). However, recent clinical trials using HDACi have been far from promising. Until now, the effect of HDACi on pancreatic cancer stroma has not been well studied. Recently, we published work demonstrating that administration of all-trans-retenoic acid (ATRA) reverses the myofibroblast/activated phenotype of pancreatic stellate cells (PSC) to a quiescent one (Froeling et al. 2011). In addition, functional genetic screens have revealed that retinoic acid signaling is a target of HDACi (Epping et al. 2007). Consequently, the tumor-stroma cross-talk may be a vital target for HDACi, which has been ignored in studies thus far. Aim: To investigate the effect of HDACi on the tumor-stroma cross talk using the 3-D organotypic collagen/matrigel culture model. Methods: Vorinostat (suberoylanilide hydroxamic acid, SAHA), valproic acid (VPA), and trichostatin acid (TsA) were used to treat PSC, Capan1, and AsPc1 cell lines. Treatment was done on cells plated both in 2-D (on plastic) and in 3-D using collagen/matrigel organotypic culture models. Results: Using HDACi on the cells in 2-D cell culture showed that PSC were more susceptible to treatment especially with low physiologic doses of HDACi. This was shown by MTS and PI DNA content FACS analysis. There was increase in sub-G1 fraction with corresponding decreased viability on MTS data, suggesting apoptosis. A corresponding dose related increase in acetylated histone 3 was shown on Western blots (a marker for effectiveness of HDACi). Similar effect was shown on cancer cells but only using higher nonphysiologic doses. In the 3-D culture model, SAHA was used to treat cancer cells co-cultured with PSC daily for 7 days. There was a dose related reduction in the number of cancer cells, and decreased invasion into the gels. This was accompanied by reduction in Ki-67 staining and increase caspase-3. There is also a corresponding dose related increased acetylated histone-3. Interestingly this effect was not as significant when cancer cells were used on their own despite similar effect on acetylated histone 3. Conclusion: Our data so far show the PSC to be a more appropriate target for HDACi than the cancer cells using physiologic doses, which can be used in-vivo. It is now established that PSC play a key role in promoting cancer cell growth and invasion. When co-cultured together in 3-D, HDACi is reversing the proliferative and metastatic effect of the PSC on the Capan1. HDACi have shown to be an interesting drug for targeting tumor-stroma cross-talk in pancreatic cancer. The machinery of the effect is currently being studied with gene expression microarray and FACS of cells isolated from the 3-D gels. Citation Format: Mohammed Ayman Ghallab, Rosignoli Guglielmo, Irene Sangrador Escrig, Ian R. Hart, Satya Bhattacharya, Hemant M. Kocher. Targeting pancreatic cancer stroma with histone deacetylase inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B80.