Fascin is regulated by slug, promotes progression of pancreatic cancer in mice, and is associated with patient outcomes.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is often lethal because it is highly invasive and metastasizes rapidly. The actin-bundling protein fascin has been identified as a biomarker of invasive and advanced PDAC and regulates cell migration and invasion in vitro. We investigated fascin expression and its role in PDAC progression in mice.
METHODS: We used KRas(G12D) p53(R172H) Pdx1-Cre (KPC) mice to investigate the effects of fascin deficiency on development of pancreatic intraepithelial neoplasia (PanIn), PDAC, and metastasis. We measured levels of fascin in PDAC cell lines and 122 human resected PDAC samples, along with normal ductal and acinar tissues; we associated levels with patient outcomes.
RESULTS: Pancreatic ducts and acini from control mice and early-stage PanINs from KPC mice were negative for fascin, but approximately 6% of PanIN3 and 100% of PDAC expressed fascin. Fascin-deficient KRas(G12D) p53(R172H) Pdx1-Cre mice had longer survival times, delayed onset of PDAC, and a lower PDAC tumor burdens than KPC mice; loss of fascin did not affect invasion of PDAC into bowel or peritoneum in mice. Levels of slug and fascin correlated in PDAC cells; slug was found to regulate transcription of Fascin along with the epithelial-mesenchymal transition. In PDAC cell lines and cells from mice, fascin concentrated in filopodia and was required for their assembly and turnover. Fascin promoted intercalation of filopodia into mesothelial cell layers and cell invasion. Nearly all human PDAC samples expressed fascin, and higher fascin histoscores correlated with poor outcomes, vascular invasion, and time to recurrence.
CONCLUSIONS: The actin-bundling protein fascin is regulated by slug and involved in late-stage PanIN and PDAC formation in mice. Fascin appears to promote formation of filopodia and invasive activities of PDAC cells. Its levels in human PDAC correlate with outcomes and time to recurrence, indicating it might be a marker or therapeutic target for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a median survival of 6 months and a 5-year survival rate of <5%. Ninety percent of patients have surgically unresectable disease at diagnosis and the majority of patients who undergo resection for localized lesions develop recurrent or metastatic disease. Consequently, the development of more effective strategies to combat metastasis is of paramount importance.

Human PDAC arises from pancreatic intraepithelial neoplasias (PanINs) frequently driven by activating mutations in KRas, followed by loss or mutation of tumor suppressors, such as p53. Pdx1-Cre−driven expression of KRasG12D and Trp53R172H in murine pancreas mimics the human disease and importantly the histopathology. Disease progression and sites of metastases also mirror the human disease, providing a good model for human PDAC.

Slug is a snail family transcription factor that orchestrates the epithelial to mesenchymal transition (EMT) during developmental programs, including in the mouse pancreas. The snail family transcription factors repress epithelial-specific genes and enhance mesenchymal-associated genes. Snail proteins bind to specific E-box sequences in promoters or introns and regulate gene expression. In the pancreas, slug (also called snail2) is expressed in a subset of pancreatic embryonic epithelial cells and is associated with endocrine cells delaminating from primitive ductal tubules and migrating into the parenchyma. Slug expression is highest in those cells of the embryonic pancreas that have lowest levels of E-cadherin, including developing islet cells. Snail family transcription factors have also been implicated in tumor progression and metastatic dissemination. EMT occurs in PDAC and is thought to be an important process in metastatic spread.9, 10

Expression of the actin bundling protein fascin is tightly regulated during development, with fascin present transiently in many embryonic tissues and later only in selected adult tissues.11, 12 The fascin-deficient mouse develops largely normally. Fascin expression is low or absent from adult epithelia, but is often highly elevated in malignant tumors (reviewed in Hashimoto et al and Machesky et al) and its overexpression is associated with poor prognosis. Fascin is enriched in cancer cell filopodia (reviewed in Hashimoto et al) and in invadopodia.14, 15 Fascin is also expressed by fibroblasts and dendritic cells and is associated with stroma.11, 12 Fascin has also been associated with metastatic spread of breast cancer and tumor self seeding. However, the effect of loss or inhibition of fascin has not been previously tested in a spontaneous tumor model to determine whether fascin impacts on tumor progression, invasion, or metastasis.

Materials and Methods

Genetically Modified Mice

All experiments were performed according to UK Home Office regulations. Mouse models are described in Supplementary Material.

Immunoblotting and Quantitative Polymerase Chain Reaction

Immunoblotting and quantitative polymerase chain reaction were carried out by standard protocols (details in Supplementary Material; n = 3 independent experiments in triplicate).

Human Tissue Analysis

The human pancreaticobiliary tissue microarray was described previously.17, 18 (see Supplementary Material). All statistical analyses were performed using SPSS software, version 15.0 (SPSS Inc, Chicago, IL). We used Oncomine to examine fascin and slug expression in Jimeno pancreas, Pei pancreas, Badea pancreas, and Wagner cell line.

Cell Culture and Expression of Small Interfering RNA or Constructs

PDAC cell lines were generated from primary pancreatic tumors from KRasG12D p53R172H Pdx1-Cre (KPC) or fascin-deficient KPC (FKPC) mice (see Supplementary Material). All experiments used cells of <6 passages. Standard methods for small interfering RNA were described previously.

Tissue Immunofluorescence

For staining fascin, slug, snail, and twist, cells were fixed with −20°C methanol for 10 minutes. For all other staining, cells were fixed in 4% formaldehyde as described previously. Primary antibodies were detected with Alexa 488, Alexa 594, and Alexa 647-conjugated secondary antibodies. Samples were examined using Olympus FV1000 or Nikon A1 inverted laser scanning confocal microscope.

Immunohistochemistry, Live Cell Imaging, and Cell Growth Assays

Standard methods were used. See Supplementary Material for details.

In Vivo PDAC Transplantation Studies

For mesenteric and diaphragm seeding experiments, 1 × 106 PDAC cells in 100 μL phosphate-buffered saline were introduced into each nude mouse (CD-1 nude females, 6 weeks old; Charles River Laboratories, Wilmington, MA) by intraperitoneal injection and tumor nodules were quantified after 2 weeks.

Results

Fascin Expression Correlates With Poor Survival and Time to Recurrence In Human PDAC

Of 122 primary human resected PDAC, fascin was absent from normal ductal and acinar tissue, but prominent in PDAC cytoplasm (Figure 1A). Ninety-five percent of human PDAC expressed fascin and a high histoscore significantly correlated with decreased overall survival (Figure 1B), high tumor grade (Figure 1C; median histoscore 104.4 vs 72.8; P < .05), and vascular invasion (Figure 1D; median histoscore 94.5 vs 62.2; P < .04). Fascin levels did not correlate with lymph node status, tumor stage, perineural invasion, and lymphatic invasion (data not shown). In a multivariate Cox proportional-hazards regression analysis, high fascin expression only reached borderline significance as an independent predictor of poor survival, with a hazard ratio of 0.663 (95% confidence interval: 0.44−1; P = .05) (Supplementary Table 1). Importantly, fascin levels strongly correlated with time to recurrence, indicating potential importance as a predictor of tumor dissemination (Figure 1E; P < .0005).

Figure 1

High fascin histoscore predicts poor survival and recurrence in human PDAC. (A) Representative images of fascin staining in human PDAC. (B) Kaplan-Meier analysis showing cases with high histoscore have poorer outcomes compared with low expression (P = .011 by log-rank test). (C) Boxplot of fascin histoscore vs tumor grade. (D) Boxplot of fascin histoscore vs vascular invasion. (E) Boxplot of fascin histoscore vs time to recur. (F) Representative fascin staining in KPC mice as indicated. Yellow dashes outline the tumor. Insets show high-magnification views of ductal cells. Purple arrows: fascin-positive cells in normal islets. Fascin-positive cells in PanIN3 are indicated by purple arrows. Scale bars = 50 μm for normal and PanINs, 200 μm for early PDAC.

To explore a functional role of fascin, we used a mouse model of pancreatic cancer (KPC mice) recapitulating both pre-invasive PanIN (grade 1−3) and invasive, metastatic PDAC. Wild-type ducts and acini and PanIN1−2 from 10-week-old KPC mice were negative for fascin (Figure 1F). Around 6% of PanIN3 and 100% of PDAC (both 10-week and advanced tumors) (Supplementary Table 2) were fascin positive (Figure 1F) and fascin was expressed in both well and poorly differentiated areas (data not shown).

Fascin null mice had normal-sized pancreata with no apparent changes in tissue structure or proliferation (Supplementary Figure 1). Although fascin is weakly expressed by a few cells in the islets of Langerhans (Figure 1F), fascin null mice had normal peripheral blood levels of several markers indicating normal pancreatic function (Supplementary Table 3). Development of PanIN in KrasG12D or KrasG12D and p53R172H expressing pancreata was not changed by loss of fascin (Supplementary Figure 2). Loss of fascin also did not affect progression, morphology, or proliferation of cells in an acute model of pancreatitis using cerulein injection (Supplementary Figure 3). However, by 21 days of cerulein treatment, fascin was detected in stroma and epithelium of PanIN of KC animals (Supplementary Figure 3). However, loss of fascin did not affect the numbers of monocytes, lymphocytes, or neutrophils recruited to acute PanINs, revealing no gross abnormalities in the immune response to PanIN in the fascin null mice (Supplementary Figure 3E and F). In summary, fascin expression was detected in a minority of PanIN3 and all PDAC and loss of fascin did not detectably affect pancreas development or PanIN.

Loss of Fascin Enhances Survival and Decreases Early Tumor Burden

Fascin expression or function has not been studied before in the context of spontaneous tumor development, so we crossed the fascin knockout mouse with KPC to make FKPC (Figure 2A). Pdx1-Cre−mediated recombination appeared normal in fascin-deficient mice (Supplementary Figure 4A), which showed a significant increase in survival (Figure 2B). Fascin was expressed in KPC and absent from FKPC tumors (Figure 2C). Fascin null mice displayed similar end-point tumor histology and mass (Figure 2D), with no significant difference in the number of undifferentiated or sarcomatoid lesions in the cohorts (not shown). KPC and FKPC tumors showed identical proportions of cell proliferation and death (Figure 2E and Supplementary Figure 4B). There was no detectable difference in recruitment of T cells (CD3), B cells (CD45R), macrophages (F4/80), or neutrophils (NIMP) between KPC and FKPC tumors (Supplementary Figure 4C and D) or difference in platelet endothelial cell adhesion molecule staining of vascularization (Supplementary Figure 4E and F). Together, these data suggest that cell proliferation, cell death, and fascin-deficient microenvironment do not contribute significantly to prolonged survival of FKPC mice.

Figure 2

Fascin is required for early PDAC formation. (A) Gene targeting strategy for generating FKPC mice. (B) Kaplan-Meier curves. (C) Top: Western blot analyses of tumor tissue. Bottom: Histology of PDAC H&E (top), immunohistochemistry for fascin (middle) and p53 (bottom). (D) Dot plot of primary tumor-to-body weight ratios at sacrifice (mean ± SEM). (E) Two-hour bromodeoxyuridine (BrdU), Ki67+, phospho-histone H3+ (PHH3), and cleaved caspase 3+ (CC3) cells in PDACs from KPC and FKPC mice. n ≥ 16 fields from n ≥ 4 mice (mean ± SEM). (F) Left: Number of PDAC-positive KPC and FKPC mice at indicated times. ∗P < .05 by χ2 test. Middle: Primary pancreas-to-body weight ratios (mean ± SEM). ∗P < .05; ∗∗P < .01 by Mann Whitney U test. Right: Relative tumor size, lower quartile, median, and upper quartile are shown. ∗P < .05 by Mann Whitney U test. Scale bars in (C) = 100 μm.

We next examined mice at earlier time points during PDAC onset and progression. No differences were found at 6 weeks (Figure 2F), but by 10 weeks, 6 of 9 KPC vs 1 of 9 FKPC mice showed tumors (Figure 2F). By 15 weeks, 9 of 10 KPC vs 3 of 6 FKPC mice showed tumors and FKPC showed smaller tumors (Figure 2F). Loss of fascin significantly delays onset of PDAC and reduces early PDAC tumor burden, a surprising effect that has not been described previously.

Slug Drives Fascin Expression in PDAC

During the development of PDAC, ductal cells undergo EMT. Fascin is principally expressed in neural and mesenchymal derivatives during mammalian embryonic development,23, 24 suggesting that fascin could be a potential EMT target. EMT involves 3 families of transcription factors, the snail, ZEB, and bHLH families.7, 25 We generated 10 independent KPC mouse PDAC cell lines that showed heterogeneous expression of E-cadherin, fascin, and EMT transcription factors (Tfs) (Figure 3A), while normal primary ductal epithelial cells did not detectably express fascin or EMT Tfs (Supplementary Figure 5A and B). Co-expression of E-cadherin and EMT Tfs indicate that most of our PDAC cell lines were in an intermediate stage of EMT (Figure 3A, Supplementary Figure 5C). Fascin-deficient PDAC cells also showed a similar heterogeneous expression of E-cadherin, fascin, and EMT Tfs (Supplementary Figure 5D). Slug, zeb1, and zeb2 were expressed in all of our PDAC cell lines, while twist and snail were expressed in a subset (Figure 3A). Levels of fascin and slug correlated most closely (Figure 3A and B). Fascin and slug expression also correlated in a dataset of 23 human pancreatic cancer cell lines (Supplementary Figure 5E).

Figure 3

Fascin is a target of slug in PDAC. (A) Expression of EMT markers in a representative panel of 10 independent KPC PDAC cell lines. (B) Spearman correlation analysis of fascin and slug protein expression in mouse PDAC cell lines. Fascin and slug expression level in PDAC cell lines was plotted as relative expression to 070669 PDAC cell line, with other cell lines numbered as in (A). (C) Left: Western blot analysis with control, Flag-slug, Flag-snail, and twist expressing 070669 PDAC cells for proteins as indicated. Bar graphs: Relative protein levels of fascin or quantitative polymerase chain reaction analysis mRNA in 070669 PDAC cells expressing EMT Tfs as indicated (mean ± SEM, n = 3). ∗P < .05; ∗∗P <.01, Student t test. (D) Phase and immunofluorescence microscopy of 070669 PDAC cells expressing EMT Tfs as indicated. Low E-cadherin and high fascin expressing cells are indicated by yellow arrows. Scale bars in (D) = 10 μm for immunofluorescence, 50 μm for phase. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loading control in (A) and (C).

Epithelial-like 070669 PDAC cells expressed a low level of fascin (Figure 3A), which increased >2-fold on Flag-slug or snail transfection (Figure 3C). Expressing snail or slug also suppressed E-cadherin but up-regulated fascin (Figure 3D and Supplementary 6A). Knockdown of slug reduced fascin expression (Supplementary Figure 6B), and stable expression of twist (Figure 3C and D and Supplementary Figure 6A) or transient knockdown of zeb1, zeb2, or E-cadherin, did not change fascin levels (Supplementary Figure 6C). Knockdown of fascin did not affect slug expression (Supplementary Figure 6C). These observations were confirmed in 061843 PDAC cells (Supplementary Figure 6D and E). Slug mediates fascin expression in PDAC cells. In addition, expression of slug or snail in human pancreatic cancer cells PANC-1 and human colon cancer cells HT29 induced fascin expression (Supplementary Figure 6F), suggesting a general effect of slug and snail on fascin expression in both mouse and human cancer cells.

Slug and Fascin Co-express During PDAC Progression

We next investigated expression of fascin and slug during EMT changes in KPC PDAC tumors. Interestingly, fascin and slug were both absent from ductal and acinar cells in normal pancreas and PanIN1/2 lesions (Figure 4A). Slug was expressed in fascin-positive (but not negative) PanIN3 lesions (Figure 4A), indicating a correlation between early markers of EMT and fascin expression during PDAC progression. Fascin and slug were present in all PDACs, regardless of E-cadherin staining or differentiation status (Figure 4B). In addition, fascin expression significantly correlated with slug expression in 3 independent cohorts of pancreatic cancer patients (Supplementary Figure 7). We propose that slug-induced EMT is an important regulator of fascin expression in pancreatic cancer.

Figure 4

Slug drives fascin expression in KRasG12D p53R172H Pdx1-Cre (KPC)-driven PDAC. (A) Fluorescent images of sections from normal pancreas and PanINs co-stained for E-cadherin (W, white), fascin (green), slug (red), and DNA (4′,6-diamidino-2-phenylindole [DAPI], blue). Insets show high-magnification views of ductal cells. (B) Well (top) and poorly differentiated (bottom) PDACs from KPC mice co-stained for E-cadherin, fascin, and slug. Insets higher magnification. Scale bars in (A) and (B) = 20 μm.

Fascin Is a Direct Slug Target Gene

Given the induction of fascin by slug and their tight association in human and mouse pancreatic cancer, we set out to determine whether fascin is a direct transcriptional target of slug. We screened the promoter and first intron region of mouse fascin for slug-binding E-box sequences (CACCTG or CAGGTG). We found a potential E-box sequence CACCTG located within the first intron of the mouse fascin gene at +2470 to +2475 bp (Figure 5A). This consensus E-box sequence is highly conserved among mammalian fascins (Figure 5A). We designed 3 sets of primers around the putative E-box sequence: primer set 1 targets the identified E-box, while primer sets 2 and 3 target adjacent regions (Figure 5A). Slug co-precipitated with the putative fascin E-box element (Figure 5B). Cotransfection of the +2345 to +2600 region of the fascin first intron in a luciferase reporter plasmid with a plasmid expressing slug into 070669 PDAC cells drove a significant increase in luciferase activity (Figure 5C). Mutagenesis of the E-box sequence eliminated the ability of slug to induce luciferase activity (Figure 5C). We propose that fascin is a direct transcriptional target of slug.

Figure 5

Fascin is a slug target gene. (A) Schematic showing the potential E-box element in intron 1 of the fascin gene and regions targeted by 3 primer pairs (#1−3, red lines). Primer pair #1 targets the putative E-box, and primer pair and #2 and #3 target downstream regions. The number in parentheses indicates distance downstream of transcription start site. (B) Chromatin from 070669 PDAC cells expressing Flag-slug was immunoprecipitated using Flag antibody and polymerase chain reaction was performed on the chromatin Immunoprecipitation product using 3 primer pairs. Primers for an E-cadherin promoter E-box element were used as positive control (mean ± SEM). ∗∗P < .01 by Student t test. (C) Top: the putative E-box on the mouse fascin gene and mutations. Bottom: Relative luciferase activities of 070669 PDAC cells transfected as indicated. n = 3 experiments (mean ± SEM). ∗∗P < .01; ∗P < .05 by Student t test.

Fascin Is Required for Local and Distant Metastasis but Not Invasion

We next explored the hypothesis that fascin was a driver of invasion and metastasis in PDAC. Invasive PDAC was present in around half of KPC mice, and this was histologically similar in FKPC mice (Figure 6A and B). More than 80% of KPC mice, but only 30% of FKPC mice, developed abdominal distension due to hemorrhagic ascites (Figure 6C). On average, KPC mice harbored 1.57 mL ascitic fluid, and FKPC mice showed almost none (Figure 6C). Metastasis was dramatically reduced in FKPC mice (Figure 6B and Supplementary Table 4). Around 95% of KPC mice and only 55% of FKPC mice had local metastasis to intestinal mesentery (Figure 6B, D, and E). Forty-four percent of KPC mice, but only 13% of FKPC mice, developed diaphragm metastasis (Figure 6B). Similar to local metastasis, 52% of KPC mice and only 13% of FKPC mice showed distant liver metastasis (Figure 6B and F). Both mesenteric and liver metastases of KPC mice were positive for fascin and p53 (Figure 6D and F). KPC mice had shorter survival overall than FKPC with liver metastases (Figure 6E). We conclude that loss of fascin significantly reduces ascites and metastasis to mesentery, diaphragm, and liver.

Figure 6

Fascin is required for efficient metastasis in KPC mice. (A) H&E staining of (left) bowel and (right) peritoneal invasion by cells from KPC and FKPC tumors. Insets show zoom of invasion area. Black arrows indicate direction of invasion. (B) Table shows incidence of invasion (top) and metastasis (bottom) in KPC and FKPC mice with PDAC (∗P < .05; ∗∗P < .01, χ2 test). (C) Incidence and volume of ascites in PDAC-bearing KPC and FKPC mice (left: ∗∗P < .01, χ2 test; right: mean ± SEM. ∗∗P < .01 by Mann-Whitney U test). (D) Top: Representative pictures of metastatic nodules (red arrows) on intestinal mesentery of KPC and FKPC mice. Bottom: Mesenteric metastasis from KPC mice H&E (left), fascin (middle), and p53 (right). (E) Left: Number of mesenteric metastases; ∗∗P < .01 by Mann-Whitney U test. Right: Survival of mice with liver metastases. Blue and red dotted lines indicate median survival for KPC and FKPC mice, respectively. (F) Liver metastasis from KPC mice for H&E (left), fascin (middle), and p53 (right). Scale bar in (A) and (D) = 100 μm and in (F) = 50 μm.

Fascin Mediates Peritoneal Metastasis Via Promotion of Transmesothelial Migration

To further investigate the mechanism by which fascin promotes metastasis, we first examined the actin dynamics of PDAC cells (105768) from the FKPC mice compared with the same cell line rescued with GFP-fascin. GFP-fascin concentrated in filopodia (Figure 7A and Video 1). Fascin rescue cells showed dynamic filopodia assembly and turnover (Supplementary Figure 8A and B). Filopodia were significantly less frequent, shorter, and shorter-lived in fascin-deficient cells than fascin-rescued cells (Supplementary Figure 8B). Lamellipodial dynamics were greater in fascin-rescued cells (Supplementary Figure 8C and Video 2). Expression of fascin significantly enhanced protrusion frequency, distance protruded, and protrusion rate, and decreased protrusion persistence (Supplementary Figure 8C). Fascin-rescued PDAC cells migrated faster than fascin-deficient cells (Supplementary Figure 8D and Video 3). Fascin-expression status did not affect growth in 2D or 3D (Supplementary Figure 8E), similar to PDAC in vivo. In addition, fascin-rescued cells behaved similarly to fascin-deficient cells during anoikis (Supplementary Figure 8E). Fascin expression increases PDAC cell migration via lamellipodial and filopodial dynamics, but does not affect growth and survival.

Figure 7

Fascin mediates peritoneal metastasis via promotion of transmesothelial intercalation. (A) Still image of live GFP-fascin in PDAC cells transmigrating through a red CMTPX CellTracker (CT)-labeled confluent Met5A monolayer. Yellow stars indicate fascin-positive filopodia. (B) Intercalation for individual cells during 10 hours (n = 100 cells, 8 fields, mean ± SEM, n = 3) ∗∗P < .01 by Student t test. (C) Time-lapse video stills of PDAC cells intercalating between MCs. Protrusions are indicated by yellow arrows. (D) PDAC cells as indicated were injected intraperitoneally into nude mice. Yellow arrows indicate tumor nodules. (E) Mesenteric tumor nodules from GFP-fascin−rescued cells express fascin and p53. Insets show high magnification. (F) Number of mesenteric and diaphragm metastases. n = 10 mice per condition. ∗P < .05; ∗∗P < .01 by Mann-Whitney U test. Scale bars in (A), (C), and (D) = 10 μm and in (E) = 100 μm.

Formation of mesenteric and diaphragm metastases involves transmigration of cancer cells through the mesothelial cell (MC) layer.27, 28 We tested a potential role for fascin in mesothelial transmigration by plating PDAC cells (105768) on top of a monolayer of human Met5a MCs. PDAC cells opened MC junctions and intercalated themselves between MCs (Supplementary Figure 9A). GFP-fascin localized intensively to the filopodia at the leading edge of transmigrating PDAC cells (Figure 7A and Video 4). About 75% of fascin-rescued PDAC cells, but only 35% of fascin-deficient cells, intercalated by 10 hours (Figure 7B, Supplementary Figure 9B, and Video 5). Fascin knockdown in KPC 070669 PDAC cells also significantly reduced intercalation (Supplementary Figure 9C–E). GFP-fascin−rescued cells generated protrusions that more effectively transmigrated than fascin nulls (Figure 7C and Video 6). Nude mice injected with fascin-deficient PDAC cells developed significantly fewer mesenteric or diaphragm metastatic foci than those with fascin-rescued cells (Figure 7E and F). These results are consistent with our spontaneous mouse model and suggest that targeting the interaction of PDAC cells with the mesothelium through fascin depletion is sufficient to reduce metastasis in vivo.

Discussion

Fascin Is an EMT Target in Pancreatic Cancer

Nearly all human PDAC expressed fascin, and a higher fascin histoscore correlated with poor outcomes, vascular invasion, and time to recurrence. Similar correlations have been reported for hepatocellular and extrahepatic bile duct carcinomas.29, 30 Fascin expression in smaller cohorts of human PDAC and PanIN,31, 32 and also in pancreatobiliary adenocarcinomas and pancreatic intraductal papillary mucinous carcinoma, correlated with shorter survival times and more advanced stages. Fascin expression contributes to progression of human PDAC, but is only of borderline significance as a prognostic indicator, indicating that other factors contribute to recurrence and spread.

Fascin is a wnt target in colorectal cancer, where it localizes to tumor invasive fronts but is down-regulated in metastases. However, in KrasG12D- and p53R172H-driven pancreatic cancer, fascin is evenly expressed in tumors and remains highly expressed in liver and peritoneal metastases. Unlike colorectal cancer, the role of wnt signaling in pancreatic cancer progression is less clear, and we find that fascin is an EMT target of the Tf slug. Slug is expressed in pancreatic endocrine progenitor cells and effects EMT changes and migration during early embryonic development. We speculate that PDAC cells might reacquire slug and fascin during a partial reversion to an embryonic migratory state.

Fascin Contributes to PDAC Progression

There is controversy about whether gene changes that confer metastatic dissemination of pancreatic cancer (or other cancers) occur early in tumor formation or later. A recent study provided compelling evidence based on lineage tracing of cells by tumor mutation analysis that metastasis could occur even before there was a recognizable tumor. Our finding that fascin expression happens during late PanIN to PDAC transition suggest that EMT changes that promote metastasis start to happen early. EMT has been correlated with tumor-initiating (stem) cell properties and as a part of an EMT program. Fascin expression might allow tumor stem cells to thrive during initial tumor formation, as well as later during metastasis. Perhaps primary tumors and metastases first arise from small nests of fascin-positive cells in PanIN3. In this case, expression of fascin in PanINs might be predictive of tumor formation and metastasis.

Fascin in Invasion and Metastatic Colonization

Fascin is not only expressed in PDAC tumor cells, but also in stroma of PDAC and of some PanIN. Because our fascin knockout is global and constitutive, loss of fascin in the stroma might have contributed to the phenotypes we observed. However, we could not detect any gross changes in the stromal immune cell component or blood vessel density of fascin knockout tumors, and we recently reported that fascin loss is dispensable for growth of transplanted tumors.

Fascin has been implicated in migration and invasion in vitro, so it was surprising that fascin loss had no effect on invasion in vivo. We previously observed that only melanoma cell lines displaying elongated mesenchymal mechanisms of invasion were dependent on fascin. Collective invasion into bowel or peritoneal wall is not limited by loss of fascin and might also not be limited by matrix remodeling or invadopodia formation. Collective PDAC invasion could occur in physiological clefts between tightly packed collagen bundles or muscle strands, and fascin-mediated protrusions might not be crucial.

We show that fascin null cells are less able to colonize the mesentery. Rho-associated colied-coil-containing protein kinase and myosin-mediated contractility are required for transmesothelial migration of human multiple myeloma and ovarian cancer cells.40, 41 We provide mechanistic evidence that fascin drives long filopodia that cross between the mesothelial cells and make initial contact with the substratum to aid transmigration. Our study suggests that, at least for PDAC, it is not invasion of the primary tumor, but rather colonization of the new site that is most affected by fascin loss.