LIV-1 Promotes Prostate Cancer Epithelial-to-Mesenchymal Transition and Metastasis through HB-EGF Shedding and EGFR-Mediated ERK Signaling

LIV-1, a zinc transporter, is an effector molecule downstream from soluble growth factors. This protein has been shown to promote epithelial-to-mesenchymal transition (EMT) in human pancreatic, breast, and prostate cancer cells. Despite the implication of LIV-1 in cancer growth and metastasis, there has been no study to determine the role of LIV-1 in prostate cancer progression. Moreover, there was no clear delineation of the molecular mechanism underlying LIV-1 function in cancer cells. In the present communication, we found increased LIV-1 expression in benign, PIN, primary and bone metastatic human prostate cancer. We characterized the mechanism by which LIV-1 drives human prostate cancer EMT in an androgen-refractory prostate cancer cells (ARCaP) prostate cancer bone metastasis model. LIV-1, when overexpressed in ARCaPE (derivative cells of ARCaP with epithelial phenotype) cells, promoted EMT irreversibly. LIV-1 overexpressed ARCaPE cells had elevated levels of HB-EGF and matrix metalloproteinase (MMP) 2 and MMP 9 proteolytic enzyme activities, without affecting intracellular zinc concentration. The activation of MMPs resulted in the shedding of heparin binding-epidermal growth factor (HB-EGF) from ARCaPE cells that elicited constitutive epidermal growth factor receptor (EGFR) phosphorylation and its downstream extracellular signal regulated kinase (ERK) signaling. These results suggest that LIV-1 is involved in prostate cancer progression as an intracellular target of growth factor receptor signaling which promoted EMT and cancer metastasis. LIV-1 could be an attractive therapeutic target for the eradication of pre-existing human prostate cancer and bone and soft tissue metastases

Development of Trust in an Online Breast Cancer Forum: A Qualitative Study

Background: Online health forums provide peer support for a range of medical conditions, including life-threatening and terminal illnesses. Trust is an important component of peer-to-peer support, although relatively little is known about how trust forms within online health forums. Objective: The aim of this paper is to examine how trust develops and influences sharing among users of an online breast cancer forum. Methods: An interpretive qualitative approach was adopted. Data were collected from forum posts from 135 threads on nine boards on the UK charity, Breast Cancer Care (BCC). Semi-structured interviews were conducted with 14 BCC forum users. Both datasets were analysed thematically using Braun and Clarke’s [2006] approach and combined to triangulate analysis. Results: Trust operates in three dimensions, structural, relational and temporal, which intersect with each other and do not operate in isolation. The structural dimension relates to how the affordances and formal rules of the site affected trust. The relational dimension refers to how trust was necessarily experienced in interactions with other forum users: it emerged within relationships and was a social phenomenon. The temporal dimension relates to how trust changed over time and was influenced by the length of time users spent on the forum. Conclusions: Trust is a process that changes over time, and which is influenced by structural features of the forum and informal but collectively understood relational interactions among forum users. The study provides a better understanding of how the intersecting structural, relational and temporal aspects that support the development of trust facilitate sharing in online environments. These findings will help organisations developing online health forums

A novel spontaneous model of epithelial-mesenchymal transition (EMT) using a primary prostate cancer derived cell line demonstrating distinct stem-like characteristics

Cells acquire the invasive and migratory properties necessary for the invasion-metastasis cascade and the establishment of aggressive, metastatic disease by reactivating a latent embryonic programme: epithelial-to-mesenchymal transition (EMT). Herein, we report the development of a new, spontaneous model of EMT which involves four phenotypically distinct clones derived from a primary tumour-derived human prostate cancer cell line (OPCT-1), and its use to explore relationships between EMT and the generation of cancer stem cells (CSCs) in prostate cancer. Expression of epithelial (E-cadherin) and mesenchymal markers (vimentin, fibronectin) revealed that two of the four clones were incapable of spontaneously activating EMT, whereas the others contained large populations of EMT-derived, vimentin-positive cells having spindle-like morphology. One of the two EMT-positive clones exhibited aggressive and stem cell-like characteristics, whereas the other was non-aggressive and showed no stem cell phenotype. One of the two EMT-negative clones exhibited aggressive stem cell-like properties, whereas the other was the least aggressive of all clones. These findings demonstrate the existence of distinct, aggressive CSC-like populations in prostate cancer, but, importantly, that not all cells having a potential for EMT exhibit stem cell-like properties. This unique model can be used to further interrogate the biology of EMT in prostate cancer

LIV-1 is a mediator in ARCaP_E cell EMT.

<p>The role of LIV-1 was assessed by its changed expression during EMT. <b>A,</b> ARCaP_E cells were treated for 48 hours with growth factors to induce EMT. RT-PCR and Western blotting were used to show increased LIV-1 expression (left panel), and the dose responsiveness of the expression (right panel). <b>B,</b> Western blotting was used to confirm EMT-like expressional changes in the treated ARCaP_E cells. <b>C,</b> mesenchymal cell-like ARCaP_M cells were subjected to siRNA knockdown for LIV-1 expression for 48 hours. RT-PCR and Western blotting were used to detect expressional changes reflecting reversal of EMT in the treated cells. <b>D,</b> Scratch wound healing and transwell invasion assays were used to determine migratory and invasive behavior in siRNA treated ARCaP_M cells. * indicates statistical significance compared to the con1 control clone (P<0.05). <b>E,</b> ARCaP_E cells were transfected with LIV-1 expression construct. RT-PCR and western blotting were performed 48 hours after transfection to detect expressional changes reflecting EMT-like events. GAPDH served as an internal control for RT-PCR reactions, and β-actin was used as a loading control in Western blotting.</p

LIV-1 promotes prostate cancer cell metastasis.^*

<p>*LIV-1-overexpression clones (LIV#8 and LIV#14) and vector-transfected clones (con1 and con2) were intracardially inoculated to athymic mice. Incidence and sites of metastasis were followed up to 4 months by necropsy and histopathologic confirmation.</p

Validation of the antibodies to LIV-1.

<p>The produced antibodies to LIV-1 were subjected to validation for specificity. <b>A,</b> HEK293 cells transiently transfected with the LIV-1 expression construct were subjected to Western blotting analysis with the antibodies to LIV-1 (upper panel). Antibody specificity was determined by pre-absorbing the antibody with the immunizing peptide (middle panel). <b>B,</b> ARCaP_E cells were transiently transfected with the LIV-1 expression construct to overexpress LIV-1 and ARCaP_M cells with the specific siRNA to suppress LIV-1 expression. In the upper 2 panels, Western blotting was performed 48 hours later with the antibodies to LIV-1. In the lower 2 panels, these cells were examined by RT-PCR to confirm the LIV-1 expression. β-actin was used as control in Western blotting and GAPDH was used as control for RT-PCR analysis. <b>C,</b> IHC was conducted to further confirm LIV-1 Ab specificity in ARCaP_E cells transiently transfected with the LIV-1 expression construct and ARCaP_M cells transiently transfected with the specific siRNA (200 ×).</p

LIV-1 overexpression induced EMT.

<p>ARCaP_E clones overexpressing LIV-1 displayed EMT-like changes in gene expression, cellular morphology and behavior. <b>A,</b> all four LIV-1 overexpressing ARCaP_E clones showed EMT-like expressional changes as detected by Western blotting, while the two vector control clones (1 and 2) retained an epithelial cell-like expression profile. <b>B,</b> cellular morphology of the LIV-1 overexpressing cells showed marked changes from the control clones (200 ×). <b>C,</b> LIV-1 overexpressing cells (LIV#8 and LIV#14 were compared with vector control clones 1 and 2 and parental ARCaP_E and ARCaP_M cells for altered migratory capability in transwell assays. Each result is the mean ± standard deviation of a triplicate assay. <b>D,</b> the LIV-1 overexpressing #8 and #14 clones were compared with vector control clones 1 and 2 and parental ARCaP_E and ARCaP_M cells for altered invasiveness. <b>*</b> indicates statistical significance compared to the con1 control clone (<i>P</i><0.05).</p

LIV-1 expression is associated with human prostate cancer progression.

<p><b>A,</b> RT-PCR and Western blotting were used to determine LIV-1 expression in different prostate cancer cell lines. β-actin was used as control in Western blotting and GAPDH was used as control for RT-PCR analysis. <b>B,</b> representative IHC images showed increased LIV-1 expression in human prostate specimens from benign to bone metastasis (125 ×). <b>C,</b> Interval plot of LIV-1 expression is shown versus prostate cancer progression from normal/benign, PIN, primary cancer to bone metastasis. The data are shown with 95% confidence interval (n  =  number of cases analyzed). The median expression for LIV-1 in bone was significantly greater than in normal/benign, PIN, and primary cancer (<i>P</i><0.001) and in primary cancer only (P = 0.002) as analyzed by Mann-Whitney test.</p