Prostate cancer (PCa) is the second most common type of cancer in American males. If caught early, there are several effective therapies to treat this disease. However, once the cancer metastasizes to the bone, there is no cure. Therefore, there is a defined clinical need to identify drug targets to treat or prevent bone metastatic PCa. Here, we study the expression of the hyaluronidases, Hyal1 and Hyal2, in relation to their role in PCa metastasis. Hyaluronidases degrade hyaluronan (HA), a glycosaminoglycan present in the extracellular matrix. By degrading HA, these enzymes may facilitate invasion by clearing a path for cancer cells to spread, thereby facilitating metastasis to the bone. Using western blotting, Q‐PCR and substrate gel electrophoresis, we measured the expression and activity of these enzymes in PCa cell lines of different metastatic abilities. By looking at differences across the cell lines, we hope to identify whether increased expression and/or activity of any of the hyaluronidases correlates with increased metastatic capability of the PCa cells. Western blotting showed similar expression of hyaluronidases throughout PCa cell lines while Q‐PCR showed increased Hyal1 expression in C4‐2 cells. A hyaluronidase activity assay using substrate gel electrophoresis showed increased hyaluronidase activity in more aggressive cell lines. Ongoing investigations use the hyaluronidase inhibitors disodium cromoglycate, L‐ascorbic acid 6‐palmitoyl, and octyl gallate to test the effects of hyaluronidase inhibition on PCa cell invasion in a 3D HA hydrogel. This project is funded by the Milton Stetson Award.
AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO
Heterotrimeric G-proteins play diverse roles in cellular signaling and have recently been shown to regulate cellular motility and invasion via activation of the Rho GTPases, specifically through G\#945;12/13 and Gq subunits. The Rho GTPases comprise a subfamily of the Ras Superfamily of monomeric G-proteins, which act as molecular switches to regulate reorganization of the actin cytoskeleton. One member of the Rho subfamily, RhoC GTPase, is overexpressed and specifically implicated in metastasis of many tumor types. However, the molecular mechanisms by which RhoC is activated during metastasis have not been demonstrated. During a cDNA microarray screen of laser capture microdissected patient samples, normal pancreatic ductal epithelial cells were compared with chronic pancreatitis and pancreatic adenocarcinomas. Expression of the Gq class heterotrimeric G-protein, G\#945;15/16, was found to be expressed 13- and 3-fold over normal and pancreatitis, respectively in the adenocarcinomas. In the current study we examined whether this unique Gq protein could activate RhoC GTPase. As proof-of-principal we transfected NIH3T3 cells with constitutively active or dominant negative G\#945;15/16, either alone or with a wild-type RhoC GTPase. We observed changes in cell morphology and a significant increase in RhoC-mediated invasion and migration in the cells expressing constitutively active G\#945;15/16.
Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2504.
PTH enhances the mechanical loading‐induced increases in bone formation that we postulate results from PTH disruption of the actin cytoskeleton to increase the mechanosensitivity of the osteoblast. Since Ras homologous A (RhoA) GTPase activation promotes actin assembly, we examined the effects of these stimuli on RhoA activation, actin organization, and cellular stiffness. Fluid shear (12 dynes/cm 2 ) increases actin stress fiber formation (ASFF) within 15 min in MC3T3‐E1 osteoblasts that is blocked by pretreatment with 50nM PTH. Using a RhoA G‐LISA assay, we found that shear activated RhoA within 15 min of the onset of shear and that 15 min pretreatment with PTH significantly inhibited this activation. AFM quantification of the stress‐strain relationship of MC3T3‐E1 cells indicated that shear produced a 6fold increase in cellular stiffness that was blocked by PTH pretreatment. Inhibition of Rho‐dependent kinase (ROCK), an effector protein of RhoA with Y27632 blocked the shear‐induced ASFF as well as the increase in cellular stiffness. These changes in cell stiffness closely correlated with shear‐induced calcium signaling. These studies suggest that PTH inhibits shear‐induced actin organization through the RhoA‐ROCK pathway and that the resultant changes in cell stiffness play a role in the PTH‐mediated enhancement of osteoblast mechanosensitivity. Supported by HHMI and NIH/NIAMS AR043222
Abstract Inflammatory breast cancer (IBC) is a highly aggressive form of locally advanced breast cancer, distinguished from other types of breast cancer by clinical, pathologic, and molecular features. It is clinically distinguished by rapid onset of primary skin changes, including redness of the skin, nipple retraction, and peau d’ aurange. Inflammatory breast cancer accounts for approximately 6-9% of new breast cancers in the United States annually and although the number of women affected by IBC is relatively modest, the overall the 5- and 10-year disease-free survival rates are less than 45% and 20%, respectively, making IBC the most lethal form of breast cancer. Evidence has suggested a role for the phosphoinositide 3-kinase/Akt (Protein Kinase B) signaling pathway in cellular motility, invasion, and metastasis. Recent evidence has shown activation of Akt2 (PKBβ) in breast cancer and its involvement in metastasis. However, the opposite appears to be true in inflammatory breast cancer (IBC), where Akt1 is involved in cellular motility and invasion. We have demonstrated a role for RhoC in conferring a highly invasive phenotype to IBC cells and are suggesting here that Akt1 phosphorylation of RhoC leads to cellular invasion. Farnesyltransferase inhibitors (FTIs) interfere with GTPase farnesylation and activity and we have shown that FTI treatment of SUM149 IBC cells leads to inhibition of the RhoC GTPase-induced invasive phenotype of IBC. RhoC is not a direct target of FTI action, however, its relative, RhoB, is a putative FTI target. It has been shown that FTI inhibition of farnesylated RhoB (fRhoB) leads to an accumulation of geranylgeranylated RhoB (ggRhoB) in the cell, which can interfere with GTPase signals and mimics the effects of FTI in IBC cells. RhoB GTPase, particularly ggRhoB, is an intracellular trafficking protein that regulates cellular localization of Akt1, thus affecting its activation. In this present study our primary objective was to examine the role of Akt1 and RhoC phosphorylation in IBC cell invasion as well as the localization of these various molecules within IBC cells upon FTI treatment. The SUM149 inflammatory breast cancer cell line overexpresses RhoC, lacks the PTEN gene, and consequently expresses a constitutively active Akt. Using the SUM149 cell line, we demonstrate that RhoC is a reputed substrate for Akt phosphorylation. We are able to decrease phospho-RhoC levels and inhibit SUM149 cellular invasion through inhibiting Akt1 activation with commercially available Akt inhibitors and siAkt1. Through immunofluorescence and protein localization experiments we are able to see a shift of phosphorylated Akt1 from the cell cytoplasm to the nucleus upon FTI treatment. We also observe accumulation of ggRhoB over time with localization of Akt1 away from the plasma membrane. These results suggest that Akt, specifically Akt1, phosphorylation of RhoC in inflammatory breast cancer cells is required for cellular invasion. We are also able to suggest that FTI treatment of IBC cells leads to increased ggRhoB levels, which may lead to changes in localization of proteins that will affect RhoC GTPase and the metastatic IBC phenotype. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-20.
Introduction: Rap1, a member of the Ras superfamily, is thought to be important in cell adhesion and proliferation. Rap1GAP is a GTPase-activating protein which inactivates Rap1. Through gene expression profiling, we found Rap1GAP expression was significantly down-regulated in pancreatic cancer (Cancer Res 2003, 63: 2649). We sought to examine the role of Rap1GAP in pancreatic cancer development and progression. Methods: To study the functional role of Rap1GAP, the pancreatic cancer cell lines Panc1 and MiaPaCa-2 were stably transfected with Rap1GAP (R-Panc1 and R-Mia). Cell proliferation was determined by cell counting and the MTS assay, colony formation using a soft-agar assay, cell mobility and invasion using a colloidal gold assay, a Matrigel gel-coated Boyden chamber and immunofluorescent staining for focal adhesion and F-actin assembly. The effect of Rap1GAP on pancreatic tumor growth and metastasis was examined using an in vivo orthotopic pancreatic cancer model (n=6/group). Results: Rap1GAP expression was significantly reduced in primary pancreatic cancers and cell lines compared to normal pancreas (3.3 and 5.3 fold, respectively). In IHC analysis, Rap1GAP was highly expressed in pancreatic ductal cells and PanIn lesions, while >50% of pancreatic cancers had no Rap1GAP expression. Loss of Rap1GAP expression correlated with poor differentiation status. Rap1 activity in R-Panc1 and R-Mia was significantly reduced compared to control cell lines. Cell proliferation and colony formation were markedly inhibited in Rap1GAP stably transfected cell lines, as was motility and invasion, compared to controls. No focal adhesion or F-actin assembly was seen in Rap1GAP stably transfected cells after serum treatment, which was prominent in controls. In vivo studies using mice injected with R-Panc1 cells had no (2/6) or very small (4/6) tumors (mean 76.4mm3) compared to controls (mean 320.4mm3). Controls demonstrated extensive local tumor invasion and liver metastasis, not evident with R-Panc1 cells. Conclusions: Loss of RAP1GAP, not previously described in human cancers, may be involved in the progression of pancreatic cancer.
Abstract The Rho GTPases are molecular switches intimately involved in cancer metastasis through orchestrating changes in the cytoskeleton leading to motility and invasion. Previously, we demonstrated that RhoC GTPase was sufficient and required for PC-3 prostate cancer invasion. Furthermore, we demonstrated that targeted down-regulation of RhoC using either a dominant-negative RhoC or by specific shRNAs led to increased and sustained activation of Rac1 GTPase. Increased levels of active Rac1 led to morphological, molecular and phenotypic changes reminiscent of epithelial to mesenchymal transition (EMT). Additionally, we demonstrated a requirement for active Rac1 GTPase in PC-3 tumor cell diapedesis across a bone marrow endothelial cell (BMEC) layer, an important step in prostate cancer skeletal metastasis. In the current study, we analyze the specific roles of Rac1, Rac3 and RhoG GTPases in tumor cell diapadesis. Use of specific siRNAs to down-regulate the individual Rac proteins demonstrated unique roles for each in promoting cell adhesion and diapedesis. We found Rac1 to be expressed at significantly higher levels than Rac3 or RhoG. However, RhoG appears to regulate PC-3 diapadesis at levels similar to Rac1. In contrast, Rac3 does not regulate diapadesis but controls PC-3 binding to substrates and BMECs. Together, these data suggest a role for coordinated activity of the Rac GTPases during diapadesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2356.
