Cholangiocarcinoma morbidity and mortality is attributable to local invasiveness and regional lymph node and distant organ metastasis. Cholangiocarcinoma progression follows a series of sequential events that resemble wound healing reactions: local invasion resembles the epithelial migration phase involving epithelial–mesenchymal transition (EMT); colonization at distant sites resembles epithelial restitution seen during the reverse process, mesenchymal–epithelial transition (MET). In this study we compare the in vivo local and metastatic growth potential of cholangiocarcinoma cell lines with respect to expression of a novel pSTAT3-dependent, biliary epithelial cell wound healing protein, small proline-rich protein 2A (SPRR2A). SPRR2A has been associated with local aggressiveness, but decreased metastatic capabilities in other cancers. Stable SPRR2A transfection into two cholangiocarcinoma cell lines (SG231 and HuCCT-1), previously shown by us to induce permanent EMT, resulted in local aggressiveness but an inability to form metastases. In contrast, SPRR2A-negative epithelial control cells showed relatively poor local aggressiveness, but readily formed metastatic tumors. Post-intrasplenic injection cell tracking showed that: (a) mesenchymal (SPRR2A+) cells were not trapped in the liver, but were rapidly cleared through mesenteric lymph nodes and did not form metastases; whereas (b) epithelial (SPRR2A−) controls were primarily entrapped within MUC-1-associated liver “micro-infarcts” that later evolved into metastatic colonies. SPRR2A-associated tumor behavior was mimicked by MUC1 shRNA, which induced EMT and, like SPRR2A+ cells, showed reduced metastatic capabilities. Cholangiocarcinoma local invasion involves EMT processes, whereas MET and MUC1 expression promote metastasis. A better understanding of disease progression should help target treatment for this deadly neoplasm.
Small proline-rich protein 2AEpithelial-mesenchymal transitionCancerMetastasisMucin 1
Supplementary material 2 (Fig. 2) Only epithelial SG231 vector cells establish tumors in the spleen (black arrows) following post-intrasplenic injection. Real time PCR for the Yb8 Alu human repeat sequence in DNA extracted from the spleens indicates no micro-metastasis present in spleens from animals receiving mesenchymal SPRR2A cell injections. Animals were given 8 weeks for tumor development unless otherwise indicated. The presence of tumor (>10 pg DNA) relative to cell type (vector vs clone) achieved significance (p = 0.02); Fisher’s Exact Test. (# = >10 pg Alu/100 ng total DNA; * autopsied at 12 weeks post-intrasplenic injection) (TIFF 16638 kb)
Supplementary material 3 (Fig. 3) Epithelial HuCCT-1 vector cells were more successful at establishing tumors following post-intrasplenic injection than SPRR2A transfectants. Real time PCR for the Yb8 Alu human repeat sequence in DNA extracted from various organs showed that epithelial vector cells more readily established tumor burden in the liver, spleen and pancreas than mesenchymal SPRR2A cells. Animals were given 16 weeks for tumor development unless otherwise indicated. The presence of tumor (>10 pg DNA) relative to cell type (vector vs clone) achieved significance in the liver (p = 0.03) and spleen (p = 0.02); Fisher’s Exact Test. (* = >10 pg human DNA/100 ng total DNA) (TIFF 21120 kb)
Supplementary material 4 (Fig. 4) Knock-down of MUC1 in SG231 vector cells causes EMT. Epithelial SG231 vector cells (GFP+) were stably transfected with MUC1 shRNA (RFP+), resulting in epithelial-to-mesenchymal phenotypic transition and reduced E-cadherin expression. (Hsp-90α = loading control; Western blots quantified using ImageJ analysis) (TIFF 12947 kb)