Crawford, Howard C.

Significance The skin and intestinal barrier are controlled by signaling scissors, termed ADAM17 (a disintegrin and metalloprotease 17), that reside in the membrane on the surface of cells. The main purpose of these signaling scissors is to liberate growth factors from their membrane anchor, allowing them to activate their receptors, including the epidermal growth factor receptor (EGFR). The ADAM17/EGFR signaling axis is tightly regulated, yet little is known about the underlying mechanism. Here we use genetic, cell biological, and biochemical approaches to identify two membrane proteins termed iRhoms 1 and 2 (inactive Rhomboid-like proteins) as crucial upstream regulators of ADAM17-dependent EGFR signaling. This uncovers the iRhoms as attractive novel targets to treat ADAM17/EGFR-dependent diseases such as cancer.

Abstract BAFF and a proliferation-inducing ligand (APRIL), which control B cell homeostasis, are therapeutic targets in autoimmune diseases. TACI-Fc (atacicept), a soluble fusion protein containing the extracellular domain of the BAFF–APRIL receptor TACI, was applied in clinical trials. However, disease activity in multiple sclerosis unexpectedly increased, whereas in systemic lupus erythematosus, atacicept was beneficial. In this study, we show that an endogenous soluble TACI (sTACI) exists in vivo. TACI proteolysis involved shedding by a disintegrin and metalloproteinase 10 releasing sTACI from activated B cells. The membrane-bound stub was subsequently cleaved by γ-secretase reducing ligand-independent signaling of the remaining C-terminal fragment. The shed ectodomain assembled ligand independently in a homotypic way. It functioned as a decoy receptor inhibiting BAFF- and APRIL-mediated B cell survival and NF-κB activation. We determined sTACI levels in autoimmune diseases with established hyperactivation of the BAFF–APRIL system. sTACI levels were elevated both in the cerebrospinal fluid of the brain-restricted autoimmune disease multiple sclerosis correlating with intrathecal IgG production, as well as in the serum of the systemic autoimmune disease systemic lupus erythematosus correlating with disease activity. Together, we show that TACI is sequentially processed by a disintegrin and metalloproteinase 10 and γ-secretase. The released sTACI is an immunoregulator that shares decoy functions with atacicept. It reflects systemic and compartmentalized B cell accumulation and activation.

Abstract The serine/cysteine protease inhibitor SCCA1 (SERPINB3) is upregulated in many advanced cancers with poor prognosis, but there is limited information about whether it makes functional contributions to malignancy. Here, we show that SCCA1 expression promoted oncogenic transformation and epithelial–mesenchymal transition (EMT) in mammary epithelial cells, and that SCCA1 silencing in breast cancer cells halted their proliferation. SCCA1 overexpression in neu+ mammary tumors increased the unfolded protein response (UPR), IL6 expression, and inflammatory phenotypes. Mechanistically, SCCA1 induced a prolonged nonlethal increase in the UPR that was sufficient to activate NF-κB and expression of the protumorigenic cytokine IL6. Overall, our findings established that SCCA1 contributes to tumorigenesis by promoting EMT and a UPR-dependent induction of NF-κB and IL6 autocrine signaling that promotes a protumorigenic inflammation. Cancer Res; 74(21); 6318–29. ©2014 AACR.

Abstract Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer related death in the U.S. This disease has a poor prognosis due to late detection and rapid progression to metastasis. ADAM10 is a membrane bound metalloproteinase responsible for shedding bioactive molecules and inducing regulated intra-membrane proteolysis of cell surface proteins such as Notch. Both ADAM10 and Notch are highly expressed in patients with PDA, with the latter being implicated in PDA progression. We have found that genetic ablation of ADAM10 in the KrasG12D/+;Ptf1aCre/+ mouse PDA model altered tumor character to favor a cystic phenotype, mixed with low grade pancreatic intraepithelial neoplasia (PanIN) and PDA. Though ADAM10 knockout mice retain the ability to form locally invasive cancer, they live 50% longer than control mice, essentially to a full 2-year life span. The phenotypes we observe closely mimic Notch-2 knockout mice, consistent with ADAM10’s role as the primary Notch activating proteinase. Further examination revealed that ADAM10 knockout mice did not show significant liver metastasis compared to control, despite their local invasiveness. The mechanism through which ADAM 10 controls a cancer cell's ability to establish distant metastases will be explored. Citation Format: Jason C. Hall, Howard C. Crawford. Ablation of a disintegrin and metalloproteinase 10 (ADAM 10) induces significant pancreatic tumor heterogeneity and blocks cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5015. doi:10.1158/1538-7445.AM2013-5015

Abstract EGFR and EGFR ligand upregulation has been widely seen in both human chronic pancreatitis and pancreatic ductal adenocarcinoma (PDA) for many years; however the role of EGFR in PDA was poorly understood. Recently we and others have shown that genetic ablation of EGFR effectively prevents both spontaneous and cerulein induced tumorigenesis in the pancreas of the LSL-KrasG12D/+;Ptf1aCre/+ (KC) mouse model of pancreatic tumorigenesis. Pockets of EGFR upregulation localized in single acini of KC mice appear to precede pancreatic intraepithelial neoplasia (PanIN) formation in spontaneous tumorigenesis, coupled with a distinct spatially localized immune response. Ubiquitous EGFR upregulation is observed throughout the pancreas upon induction of pancreatitis in wild-type (WT) and KC mice, returning to normal levels in WT mice but continuing to increase along with the widespread tumorigenesis in cerulein treated KC mice. Comparison of cerulein treated KC mice with the EGFRf/f;LSL-KrasG12D/+;Ptf1aCre/+ mice (EKC), which are largely resistant to cerulein induced pancreatitis, offers an opportunity to identify signals in the KC mouse which could be responsible for the upregulation of EGFR and subsequent tumorigenesis. Citation Format: Christopher J. Halbrook, Kenneth K. Takeuchi, Howard C. Crawford. Regulation of EGFR in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3030. doi:10.1158/1538-7445.AM2013-3030

Abstract Pancreatic ductal adenocarcinoma (PDA) arises at the convergence of genetic alterations in KRAS with a fostering microenvironment shaped by immune cell influx and fibrotic changes; identification of the earliest tumorigenic molecular mediators evokes the proverbial chicken and egg problem. Matrix metalloproteinases (MMP) are key drivers of tumor progression that originate primarily from stromal cells activated by the developing tumor. Here, MMP3, known to be expressed in PDA, was found to be associated with expression of Rac1b, a tumorigenic splice isoform of Rac1, in all stages of pancreatic cancer. Using a large cohort of human PDA tissue biopsies specimens, both MMP3 and Rac1b are expressed in PDA cells, that the expression levels of the two markers are highly correlated, and that the subcellular distribution of Rac1b in PDA is significantly associated with patient outcome. Using transgenic mouse models, coexpression of MMP3 with activated KRAS in pancreatic acinar cells stimulates metaplasia and immune cell infiltration, priming the stromal microenvironment for early tumor development. Finally, exposure of cultured pancreatic cancer cells to recombinant MMP3 stimulates expression of Rac1b, increases cellular invasiveness, and activation of tumorigenic transcriptional profiles. Implications: MMP3 acts as a coconspirator of oncogenic KRAS in pancreatic cancer tumorigenesis and progression, both through Rac1b-mediated phenotypic control of pancreatic cancer cells themselves, and by giving rise to the tumorigenic microenvironment; these findings also point to inhibition of this pathway as a potential therapeutic strategy for pancreatic cancer. Mol Cancer Res; 12(10); 1430–9. ©2014 AACR.

In response to inflammation, pancreatic acinar cells can undergo acinar-to-ductal metaplasia (ADM), a reprogramming event that induces transdifferentiation to a ductlike phenotype and, in the context of additional oncogenic stimulation, contributes to development of pancreatic cancer. The signaling mechanisms underlying pancreatitis-inducing ADM are largely undefined. Our results provide evidence that macrophages infiltrating the pancreas drive this transdifferentiation process. We identify the macrophage-secreted inflammatory cytokines RANTES and tumor necrosis factor α (TNF) as mediators of such signaling. Both RANTES and TNF induce ADM through activation of nuclear factor κB and its target genes involved in regulating survival, proliferation, and degradation of extracellular matrix. In particular, we identify matrix metalloproteinases (MMPs) as targets that drive ADM and provide in vivo data suggesting that MMP inhibitors may be efficiently applied to block pancreatitis-induced ADM in therapy.