Type I collagen forms the main constituent of the extracellular matrix in visceral organs. We reported here that cyclophosphamide (CYP)-induced cystitis significantly increased the production of type I collagen in the inflamed bladder leading to increases in the bladder weight and the thickness of the bladder wall. The endogenous nerve growth factor (NGF) in the urinary bladder regulated type I collagen expression because the neutralizing NGF antibody attenuated cystitisinduced type I collagen up-regulation in the inflamed bladder. Neutralizing NGF antibody also subsequently reversed cystitis-induced increases in bladder weight. Further studies on the intermediate signaling pathways mediating NGF-induced type I collagen expression in the inflamed bladder during cystitis revealed that Akt, JNK, and ERK1/2 activities were increased in the inflamed bladder, whereas p38 MAPK remained unchanged. Suppression of endogenous NGF level with neutralizing NGF antibody significantly blocked the increased activity of Akt, JNK, and ERK1/2 in the inflamed bladder during cystitis. These results indicate that endogenous NGF plays an important role in the activation of Akt and MAPK in the urinary bladder and in bladder hypertrophy during cystitis.

The integral interaction of signaling components in the regulation of visceral inflammation-induced central sensitization in the spinal cord has not been well studied. Here we report that phosphoinositide 3-kinase (PI3K)-dependent Akt activation and N-Methyl-D-aspartic acid receptor (NMDAR) in lumbosacral spinal cord independently regulates the activation of cAMP response element-binding protein (CREB) in vivo in a rat visceral pain model of cystitis induced by intraperitoneal injection of cyclophosphamide (CYP). We demonstrate that suppression of endogenous PI3K/Akt activity with a potent PI3K inhibitor LY294002 reverses CYP-induced phosphorylation of CREB, however, it has no effect on CYP-induced phosphorylation of NR1 at Ser897 and Ser896; conversely, inhibition of NMDAR in vivo with MK801 fails to block CYP-induced Akt activation but significantly attenuates CYP-induced CREB phosphorylation in lumbosacral spinal cord. This novel interrelationship of PI3K/Akt, NMDAR, and CREB activation in lumbosacral spinal cord is further confirmed in an ex vivo spinal slice culture system exposed to an excitatory neurotransmitter calcitonin gene-related peptide (CGRP). Consistently we found that CGRP-triggered CREB activation can be blocked by both PI3K inhibitor LY294002 and NMDAR antagonists MK801 and D-AP5. However, CGRP-triggered Akt activation cannot be blocked by MK801 or D-AP5; vice versa, LY294002 pretreatment that suppresses the Akt activity fails to reverse CGRP-elicited NR1 phosphorylation. These results suggest that PI3K/Akt and NMDAR independently regulates spinal plasticity in visceral pain model, and target of a single pathway is necessary but not sufficient in treatment of visceral hypersensitivity.

#35 TGFb signaling synergizes with HER2 in breast cancer progression. Exogenous TGFb or expression of an activated TGFb type I receptor (Alk5T204D) in MCF10A/HER2 and BT474 cells activated PI3K/Akt and enhanced survival and migration. In both cell types, TGFb stimulated P-EGFR and P-HER2 as well as the association of p85, the regulatory subunit of PI3K, with Y1289 P-ErbB3 which, in turn, activated PI3K/Akt. RNA interference of ErbB3 or treatment with pertuzumab, an antibody that blocks HER2-mediated activation of ErbB3, blocked TGFb-induced P-ErbB3, P-Akt, and cell motility. Treatment with TGFb or expression of Alk5TD increased protein levels of secreted TGFalpha, amphiregulin, and heregulin without a change in mRNA levels. The increase on secreted ErbB ligands without a change in mRNA levels suggested increased shedding of ErbB pro-ligands by tumor necrosis factor a-converting enzyme (TACE). Transfection of siRNA against human TACE but not control siRNA abrogated TGFb-stimulated ErbB receptor phosphorylation, P-Akt, and cell invasiveness. Transfection of full-length mouse TACE but not truncated TACE lacking the cytoplasmic domain reconstituted TGFb-induced ErbB phosphorylation in cells transfected with human TACE siRNA. TGF-b increased TACE phosphorylation in both serine and threonine as measured by P-Ser and P-Thr immunoblots of TACE pull downs. Incubation of TACE with Alk5TD in the presence of ATP in vitro resulted in both Ser and Thr phosphorylation in full-length but not truncated TACE. Further, addition of TGFb or expression of Alk5TD desensitized SKBR3 and BT474 cells to trastuzumab. Treatment with the type I TGFb receptor (Alk5) small molecule inhibitor LY2109761, the PI3K inhibitor LY294002, or siRNA against ErbB3 or TACE restored the inhibitory effect of trastuzumab. Finally, we generated an active Alk5 expression signature by selecting genes that were differentially expressed between BT474/Alk5TD and BT474/vector cells. Mapping of this signature to a previously published 295-breast tumor array (van de Vijver et al. and Chang et al.) revealed 90 of 271 genes in the Alk5TD signature with a >1.5-fold change from the median expression which correlated with HER2-positive and basal-like tumors and conferred a worse recurrence-free (RFS) and overall survival (OS) (RFS p=0.05, OS p=0.01). Hierarchical clustering analysis to an array data set reported by Harris et al. in 22 patients with HER2+ breast cancer treated with neoadjuvant trastuzumab and vinorelbine (Clin. Cancer Res. 2007) showed that the Alk5TD signature correlated with tumors that did not respond clinically. These data suggest that TGFb potentiates signaling downstream ErbB receptors via TACE, ErbB3, and PI3K, thus contributing to tumor progression and resistance to anti-HER2 therapies. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 35.

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