Stuart Morton

Platelet‐derived growth factor‐D and Rho GTPases regulate recruitment of cancer‐associated fibroblasts in cholangiocarcinoma

Cholangiocarcinoma (CCA) is characterized by an abundant stromal reaction. Cancer‐associated fibroblasts (CAFs) are pivotal in tumor growth and invasiveness and represent a potential therapeutic target. To understand the mechanisms leading to CAF recruitment in CCA, we studied (1) expression of epithelial‐mesenchymal transition (EMT) in surgical CCA specimens and CCA cells, (2) lineage tracking of an enhanced green fluorescent protein (EGFP)‐expressing human male CCA cell line (EGI‐1) after xenotransplantation into severe‐combined‐immunodeficient mice, (3) expression of platelet‐derived growth factors (PDGFs) and their receptors in vivo and in vitro, (4) secretion of PDGFs by CCA cells, (5) the role of PDGF‐D in fibroblast recruitment in vitro, and (6) downstream effectors of PDGF‐D signaling. CCA cells expressed several EMT biomarkers, but not alpha smooth muscle actin (α‐SMA). Xenotransplanted CCA masses were surrounded and infiltrated by α‐SMA‐expressing CAFs, which were negative for EGFP and the human Y‐probe, but positive for the murine Y‐probe. CCA cells were strongly immunoreactive for PDGF‐A and ‐D, whereas CAFs expressed PDGF receptor (PDGFR)β. PDGF‐D, a PDGFRβ agonist, was exclusively secreted by cultured CCA cells. Fibroblast migration was potently induced by PDGF‐D and CCA conditioned medium and was significantly inhibited by PDGFRβ blockade with Imatinib and by silencing PDGF‐D expression in CCA cells. In fibroblasts, PDGF‐D activated the Rac1 and Cdc42 Rho GTPases and c‐Jun N‐terminal kinase (JNK). Selective inhibition of Rho GTPases (particularly Rac1) and of JNK strongly reduced PDGF‐D‐induced fibroblast migration. Conclusion: CCA cells express several mesenchymal markers, but do not transdifferentiate into CAFs. Instead, CCA cells recruit CAFs by secreting PDGF‐D, which stimulates fibroblast migration through PDGFRβ and Rho GTPase and JNK activation. Targeting tumor or stroma interactions with inhibitors of the PDGF‐D pathway may offer a novel therapeutic approach. (Hepatology 2013;53:1042–1053)

Protein kinase A-dependent pSer(675) -β-catenin, a novel signaling defect in a mouse model of congenital hepatic fibrosis.

Genetically determined loss of fibrocystin function causes congenital hepatic fibrosis (CHF), Caroli disease (CD), and autosomal recessive polycystic kidney disease (ARPKD). Cystic dysplasia of the intrahepatic bile ducts and progressive portal fibrosis characterize liver pathology in CHF/CD. At a cellular level, several functional morphological and signaling changes have been reported including increased levels of 3′‐5′‐cyclic adenosine monophosphate (cAMP). In this study we addressed the relationships between increased cAMP and β‐catenin. In cholangiocytes isolated and cultured from Pkhd1del4/del4 mice, stimulation of cAMP/PKA signaling (forskolin 10 μM) stimulated Ser675‐phosphorylation of β‐catenin, its nuclear localization, and its transcriptional activity (western blot and TOP flash assay, respectively) along with a down‐regulation of E‐cadherin expression (immunocytochemistry and western blot); these changes were inhibited by the PKA blocker, PKI (1 μM). The Rho‐GTPase, Rac‐1, was also significantly activated by cAMP in Pkhd1del4/del4 cholangiocytes. Rac‐1 inhibition blocked cAMP‐dependent nuclear translocation and transcriptional activity of pSer675‐β‐catenin. Cell migration (Boyden chambers) was significantly higher in cholangiocytes obtained from Pkhd1del4/del4 and was inhibited by: (1) PKI, (2) silencing β‐catenin (siRNA), and (3) the Rac‐1 inhibitor NSC 23766. Conclusion: These data show that in fibrocystin‐defective cholangiocytes, cAMP/PKA signaling stimulates pSer675‐phosphorylation of β‐catenin and Rac‐1 activity. In the presence of activated Rac‐1, pSer675‐β‐catenin is translocated to the nucleus, becomes transcriptionally active, and is responsible for increased motility of Pkhd1del4/del4 cholangiocytes. β‐Catenin‐dependent changes in cell motility may be central to the pathogenesis of the disease and represent a potential therapeutic target. (Hepatology 2013;58:1713–1723)

Isolation and characterization of biliary epithelial and stromal cells from resected human cholangiocarcinoma: a novel in vitro model to study tumor-stroma interactions

Cholangiocarcinoma (CCA) is a devastating malignancy arising from the bile ducts. Cancer-associated fibroblasts (CAFs) are key players in CCA invasiveness and in the generation of a desmoplastic reaction. The aim of the present study was to develop a novel model by which to study tumor-stroma interactions using primary cultures of human biliary epithelial cells (hBECs) and stromal cells (SCs) in CCA. hBECs and SCs, isolated from surgical resections (n=10), were semi-purified by centrifugation on a Percoll gradient; hBECs were further immunopurified. hBECs and SCs were characterized using epithelial [cytokeratin 7 (CK7) and CK19] and mesenchymal [vimentin (VMN), α-smooth muscle actin (α-SMA), CD68] cell markers. The purity of cultured cells was assessed by fluorescent immunocytochemistry. hBECs were HEA125/CK7/CK19-positive and VMN/α-SMA-negative. SCs were VMN/α-SMA-positive and CK7/CK19-negative. CCA 2-D culture models have been described but they use long-standing CCA cell lines of various biliary tumor cell origins with stromal cells derived from non-cholangiocarcinoma tissues. Recently, a novel 3-D organotypic co-culture model of rat cholangiocarcinoma was described. In the present study, we obtained pure and stable primary cultures of hBECs and SCs from CCA surgical specimens. These cell cultures may provide a useful tool by which to study CCA tumor-stroma interactions.

Leukemia inhibitory factor protects cholangiocarcinoma cells from drug-induced apoptosis via a PI3K/AKT-dependent Mcl-1 activation

Cholangiocarcinoma is an aggressive, strongly chemoresistant liver malignancy. Leukemia inhibitory factor (LIF), an IL-6 family cytokine, promotes progression of various carcinomas. To investigate the role of LIF in cholangiocarcinoma, we evaluated the expression of LIF and its receptor (LIFR) in human samples. LIF secretion and LIFR expression were assessed in established and primary human cholangiocarcinoma cell lines. In cholangiocarcinoma cells, we tested LIF effects on proliferation, invasion, stem cell-like phenotype, chemotherapy-induced apoptosis (gemcitabine+cisplatin), expression levels of pro-apoptotic (Bax) and anti-apoptotic (Mcl-1) proteins, with/without PI3K inhibition, and of pSTAT3, pERK1/2, pAKT. LIF effect on chemotherapy-induced apoptosis was evaluated after LIFR silencing and Mcl-1 inactivation. Results show that LIF and LIFR expression were higher in neoplastic than in control cholangiocytes; LIF was also expressed by tumor stromal cells. LIF had no effects on cholangiocarcinoma cell proliferation, invasion, and stemness signatures, whilst it counteracted drug-induced apoptosis. Upon LIF stimulation, decreased apoptosis was associated with Mcl-1 and pAKT up-regulation and abolished by PI3K inhibition. LIFR silencing and Mcl-1 blockade restored drug-induced apoptosis. In conclusion, autocrine and paracrine LIF signaling promote chemoresistance in cholangiocarcinoma by up-regulating Mcl-1 via a novel STAT3- and MAPK-independent, PI3K/AKT-dependent pathway. Targeting LIF signaling may increase CCA responsiveness to chemotherapy.

PKA dependent p-Ser-675β-catenin, a novel signaling defect in a mouse model of Congenital Hepatic Fibrosis

Genetically determined loss of fibrocystin function causes congenital hepatic fibrosis (CHF), Caroli disease (CD), and autosomal recessive polycystic kidney disease (ARPKD). Cystic dysplasia of the intrahepatic bile ducts and progressive portal fibrosis characterize liver pathology in CHF/CD. At a cellular level, several functional morphological and signaling changes have been reported including increased levels of 3′‐5′‐cyclic adenosine monophosphate (cAMP). In this study we addressed the relationships between increased cAMP and β‐catenin. In cholangiocytes isolated and cultured from Pkhd1del4/del4 mice, stimulation of cAMP/PKA signaling (forskolin 10 μM) stimulated Ser675‐phosphorylation of β‐catenin, its nuclear localization, and its transcriptional activity (western blot and TOP flash assay, respectively) along with a down‐regulation of E‐cadherin expression (immunocytochemistry and western blot); these changes were inhibited by the PKA blocker, PKI (1 μM). The Rho‐GTPase, Rac‐1, was also significantly activated by cAMP in Pkhd1del4/del4 cholangiocytes. Rac‐1 inhibition blocked cAMP‐dependent nuclear translocation and transcriptional activity of pSer675‐β‐catenin. Cell migration (Boyden chambers) was significantly higher in cholangiocytes obtained from Pkhd1del4/del4 and was inhibited by: (1) PKI, (2) silencing β‐catenin (siRNA), and (3) the Rac‐1 inhibitor NSC 23766. Conclusion: These data show that in fibrocystin‐defective cholangiocytes, cAMP/PKA signaling stimulates pSer675‐phosphorylation of β‐catenin and Rac‐1 activity. In the presence of activated Rac‐1, pSer675‐β‐catenin is translocated to the nucleus, becomes transcriptionally active, and is responsible for increased motility of Pkhd1del4/del4 cholangiocytes. β‐Catenin‐dependent changes in cell motility may be central to the pathogenesis of the disease and represent a potential therapeutic target. (Hepatology 2013;58:1713–1723)

Protein kinase a-dependent pSer675-β-catenin, a novel signaling defect in a mouse model of congenital hepatic fibrosis: Hepatology, Vol. 00, No. X, 2013

Genetically determined loss of fibrocystin function causes congenital hepatic fibrosis (CHF), Caroli disease (CD), and autosomal recessive polycystic kidney disease (ARPKD). Cystic dysplasia of the intrahepatic bile ducts and progressive portal fibrosis characterize liver pathology in CHF/CD. At a cellular level, several functional morphological and signaling changes have been reported including increased levels of 3′‐5′‐cyclic adenosine monophosphate (cAMP). In this study we addressed the relationships between increased cAMP and β‐catenin. In cholangiocytes isolated and cultured from Pkhd1del4/del4 mice, stimulation of cAMP/PKA signaling (forskolin 10 μM) stimulated Ser675‐phosphorylation of β‐catenin, its nuclear localization, and its transcriptional activity (western blot and TOP flash assay, respectively) along with a down‐regulation of E‐cadherin expression (immunocytochemistry and western blot); these changes were inhibited by the PKA blocker, PKI (1 μM). The Rho‐GTPase, Rac‐1, was also significantly activated by cAMP in Pkhd1del4/del4 cholangiocytes. Rac‐1 inhibition blocked cAMP‐dependent nuclear translocation and transcriptional activity of pSer675‐β‐catenin. Cell migration (Boyden chambers) was significantly higher in cholangiocytes obtained from Pkhd1del4/del4 and was inhibited by: (1) PKI, (2) silencing β‐catenin (siRNA), and (3) the Rac‐1 inhibitor NSC 23766. Conclusion: These data show that in fibrocystin‐defective cholangiocytes, cAMP/PKA signaling stimulates pSer675‐phosphorylation of β‐catenin and Rac‐1 activity. In the presence of activated Rac‐1, pSer675‐β‐catenin is translocated to the nucleus, becomes transcriptionally active, and is responsible for increased motility of Pkhd1del4/del4 cholangiocytes. β‐Catenin‐dependent changes in cell motility may be central to the pathogenesis of the disease and represent a potential therapeutic target. (Hepatology 2013;58:1713–1723)

Evaluation of a new automated spotter style exam for assessment of anatomical knowledge

Skeletal material is commonly used for teaching anatomy. However, these collections are rarely used for primary research, despite their potential for student projects or more detailed anatomical investigations. Although the undocumented nature of the specimens can arguably limit study, archeologists characteristically deal with unknown skeletal material. Many methods exist to estimate age, sex, and ethnicity that could enable research. Accordingly, 94 skeletal individuals from Southampton University’s Anatomy department were assessed to determine the usability of methods routinely used in archeology. Age was estimated from degenerative joint changes and dental wear. Sexually dimorphic regions of the skull and pelvis were examined. Ethnicity was identified through craniometrics and CRANID software. As skeletons were complete, aging and sexing methods were easily applied: 95% of individuals were sexed confidently. Although all individuals were aged, often only wide estimates were produced (e.g., 21–45 years). Ethnicity however was problematic and produced less usable results. Ancestry was not determined for 19 skulls. CRANID therefore requires extremely accurate cranial measurements by a practiced researcher. This study demonstrates that archeological methods benefit anatomical research but should be selected with limitations considered. Additionally, anatomical collections are valuable teaching and research resources to osteoarcheologists who are normally limited to fragmented remains.