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The Ras/RAF/MEK/ERK pathway is an essential signaling cascade for various refractory cancers, such as those with mutant KRAS (mKRAS) and BRAF (mBRAF). However, there are unsolved ambiguities underlying mechanisms for this growth signaling thereby creating therapeutic complications. This study shows that a vital component of the pathway CRAF is directly impacted by an end product of the cascade, glutathione transferases (GST) P1 (GSTP1), driving a previously unrecognized autocrine cycle that sustains proliferation of mKRAS and mBRAF cancer cells, independent of oncogenic stimuli. The CRAF interaction with GSTP1 occurs at its N-terminal regulatory domain, CR1 motif, resulting in its stabilization, enhanced dimerization, and augmented catalytic activity. Consistent with the autocrine cycle scheme, silencing GSTP1 brought about significant suppression of proliferation of mKRAS and mBRAF cells in vitro and suppressed tumorigenesis of the xenografted mKRAS tumor in vivo. GSTP1 knockout mice showed significantly impaired carcinogenesis of mKRAS colon cancer. Consequently, hindering the autocrine loop by targeting CRAF/GSTP1 interactions should provide innovative therapeutic modalities for these cancers.

Significance A strategy to overcome therapeutic obstacles of mKRAS and mBRAF cancers is devised based on the finding, here, that the RAF/MEK/ERK cascade is by-passed by an autocrine signal loop established by interaction of CRAF with GSTP1. The interaction evokes stabilization of CRAF from proteosomal degradation and facilitation of RAF-dimer formation. Thus, blocking CRAF/GSTP1 interactions should generate additive antiproliferative effects., The Ras/RAF/MEK/ERK pathway is an essential signaling cascade for various refractory cancers, such as those with mutant KRAS (mKRAS) and BRAF (mBRAF). However, there are unsolved ambiguities underlying mechanisms for this growth signaling thereby creating therapeutic complications. This study shows that a vital component of the pathway CRAF is directly impacted by an end product of the cascade, glutathione transferases (GST) P1 (GSTP1), driving a previously unrecognized autocrine cycle that sustains proliferation of mKRAS and mBRAF cancer cells, independent of oncogenic stimuli. The CRAF interaction with GSTP1 occurs at its N-terminal regulatory domain, CR1 motif, resulting in its stabilization, enhanced dimerization, and augmented catalytic activity. Consistent with the autocrine cycle scheme, silencing GSTP1 brought about significant suppression of proliferation of mKRAS and mBRAF cells in vitro and suppressed tumorigenesis of the xenografted mKRAS tumor in vivo. GSTP1 knockout mice showed significantly impaired carcinogenesis of mKRAS colon cancer. Consequently, hindering the autocrine loop by targeting CRAF/GSTP1 interactions should provide innovative therapeutic modalities for these cancers.

Background and objective Fibrosis associated with chronic pancreatitis is an irreversible lesion that can disrupt pancreatic exocrine and endocrine function. Currently, there are no approved treatments for this disease. We previously showed that siRNA against collagen-specific chaperone protein gp46, encapsulated in vitamin A-coupled liposomes (VA-lip-siRNAgp46), resolved fibrosis in a model of liver cirrhosis. This treatment was investigated for pancreatic fibrosis induced by dibutyltin dichloride (DBTC) and cerulein in rats. Methods Specific uptake of VA-lip-siRNAgp46, conjugated with 6′-carboxyfluorescein (FAM) by activated pancreatic stellate cells (aPSCs), was analysed by fluorescence activated cell sorting (FACS). Intracellular distribution of VA-lip-siRNAgp46-FAM was examined by fluorescent microscopy. Suppression of gp46 expression by VA-lip-siRNAgp46 was assessed by immunoblotting. Collagen synthesis in aPSCs was assayed by dye-binding. Specific delivery of VA-lip-siRNAgp46 to aPSCs in DBTC rats was verified following intravenous VA-lip-siRNA-FAM and 3 H-VA-lip-siRNAgp46. The effect of VA-lip-siRNA on pancreatic histology in DBTC- and cerulein-treated rats was determined by Azan-Mallory staining and hydroxyproline content. Results FACS analysis revealed specific uptake of VA-lip-siRNAgp46-FAM through the retinol binding protein receptor by aPSCs in vitro. Immunoblotting and collagen assay verified knockdown of gp46 and suppression of collagen secretion, respectively, by aPSCs after transduction of VA-lip-siRNAgp46. Specific delivery of VA-lip-siRNAgp46 to aPSCs in fibrotic areas in DBTC rats was confirmed by fluorescence and radioactivity 24 h after the final injection. 10 systemic VA-lip-siRNAgp46 treatments resolved pancreatic fibrosis, and suppressed tissue hydroxyproline levels in DBTC- and cerulein-treated rats. Conclusion These data suggest the therapeutic potential of the present approach for reversing pancreatic fibrosis.

Background and objectives Mechanism of regeneration of remnant pancreas after partial pancreatectomy (PX) is still unknown. In this study, effect of siRNA against the collagen specific chaperone, HSP47, which inhibits collagen secretion from activated pancreas stellate cells (aPSCs), and induces their apoptosis, on regeneration of remnant pancreas was determined. Methods Pancreatectomy was performed according to established methods. Proliferation of cells was assessed by BrdU incorporation. Immunostaining of HSP47 was employed to identify PSCs. Progenitor cells were identified by SOX9 staining. Acinar cells were immunostained for amylase. Co-culture of acinar cells with aPSCs were carried out in a double chamber with a cell culture insert. siRNA HSP47 encapsulated in vitamin A-coupled liposome (VA-lip siRNA HSP47) was delivered to aPSCs by iv injection. Results In remnant pancreas of 90% PX rat, new areas of foci were located separately from duodenal areas with normal pancreatic features. After PX, BrdU uptake of acinar cells and islet cells significantly increased, but was suppressed by treatment with VA-lip siRNA HSP47. BrdU uptake by acinar cells was augmented by co-culturing with aPSCs and the augmentation was nullified by siRNA HSP47. BrdU uptake by progenitor cells in foci area was slightly enhanced by the same treatment. New area which exhibited intermediate features between those of duodenal and area of foci, emerged after the treatment. Conclusion aPSCs play a crucial role in regeneration of remnant pancreas, proliferation of acinar and islet cells after PX through the activity of secreted collagen. Characterization of new area emerged by siRNA HSP47 treatment as to its origin is a future task.

Background: Mutated KRAS (mKRAS) is the most commonly found oncogene which may play a role in carcinogenesis at relatively early stage. Although Raf-1/MEK/ERK pathway has been well characterized as a proliferative signal at downstream of mKRAS, other signals related to senescence or apoptosis were also found at downstream of mKRAS and thus mKRAS function is still needed to be explored at a molecular level. We have hitherto disclosed a close correlation of GST-π expression and mKRAS in cancer tissues, induction of GST-π expression by KRAS transfection (Gastroenterology121:865-874, 2001) and significant reduction of mKRAS positive colon polyps in GST-π KO mice (unpublished observation), suggesting some cooperative function of GST-π for mKRAS. Objective: In the present study, we attempted to clarify the molecular mechanisms of the cooperative function of GST-π for mKRAS activity in tumor cells. Results: We first intended to confirm the correlation between GST-π expression and KRAS mutation in various human cancer cell lines by Western blotting. Those cells with no mKRAS (HepG2, HeLa, MCF7) were negative for GST-π expression while mKRAS-expressing cells (PANC-1, A549, M7609) were positive for GST-π. We then using M7609 cells, examined the effect of GST-π siRNA on their growth and found a significant impairment of cell proliferation. When GST-π KD M7609 cells were examined under electron microscopy, 40% of cells showed intracytoplasmic vesicles. The vesicles were positive for LC3 punctate signal by immunofluorescent staining and showed a characteristic feature of autophagosome, an organelle enclosed by a double lipid bilayer, by electron microscopy. In these cells, phosphorylation of Akt and EGFR was substantially reduced. Since interaction of GST-π with EGFR has been reported, it is plausible that this interaction positively regulates EGFR/class I PI3K/Akt/mTOR signal which in turn inhibits autophagy formation. Another mKRAS related signal: Raf-1/MEK/ERK was also explored in its relation to GST-π function. In M7609 KD cells, both Raf-1 protein and p-Raf-1 (S338), active form of Raf-1, were significantly reduced and phosphorylation of S621 residue which is known to play a crucial role in prevention of ubiquitination of Raf-1 protein was suppressed. In these cells, phosphorylation of downstream kinases of MEK and ERK were also reduced though their total protein levels were unchanged. When the cells were treated with proteasome inhibitor (MG132), recovery of Raf-1 and p-Raf-1 protein was observed. In parental M7609 cells, GST-π was found to be preferentially bound to p-Raf-1 on co-immunoprecipitation analysis, indicating that GST-π forms complex with Raf-1to prevent the Raf-1 from proteasomal degradation. Conclusion: GST-π, on one hand prevents the cancer cells from autophagy formation and on the other hand, stimulates Raf-1/MEK/ERK signal by stabilizing Raf-1 protein in mKRAS cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1065. doi:10.1158/1538-7445.AM2011-1065