Oncogene (2004) 23, 465–473 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc CEACAM6 gene silencing impairs anoikis resistance and in vivo metastatic ability of pancreatic adenocarcinoma cells Mark S Duxbury1, Hiromichi Ito1, Michael J Zinner1, Stanley W Ashley1 and Edward E Whang*,1 1Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA Anoikis is the apoptotic response induced in normal cells maintain tissue order within multisystem organisms by by inadequate or inappropriate adhesion to substrate. It is preventingectopic cellular proliferation (Meredith et al., postulated that resistance to anoikis facilitates tumorigen- 1993; Frisch and Francis, 1994), resistance to anoikis, a esis and metastasis. Carcinoembryonic antigen-related property of tumor cells, is reported to contribute to cell adhesion molecule 6 (CEACAM6) is an immunoglo- primary tumorigenesis and metastasis in a number of bulin superfamily member overexpressed in a number of malignancies (Yawata et al., 1998; Streuli and Gilmore, human cancers and implicated in anoikis resistance. We 1999; Shanmugathasan and Jothy, 2000). Malignant tested the effect of CEACAM6 gene silencing on anoikis cells can be detected in compartments such as the blood in pancreatic adenocarcinoma cell lines. Anoikis was and peritoneal fluid, even in the early stages of disease, induced in PANC1, Capan2, MiaPaCa2 and MiaAR (a but metastasis is an inefficient process with only a small MiaPaCa2-derived anoikis-resistant subline) by culture in percentage of disseminated cells surviving to establish poly-2-hydroxyethylmethacrylate-coated wells. Anoikis metastases (Fidler et al., 1978). Inherent or acquired was quantified by YO-PRO-1/propidium iodide staining anoikis resistance is postulated to confer a selective and flow cytometry. The role of caspase activation was advantageon disseminatingtumor cells, promotingtheir determined using fluorometric profiling and the caspase survival in the absence of normal matrix attachment and inhibitor Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD- allowingthem to form metastatic colonies (Arvelo and fmk). CEACAM6 expression was suppressed by RNA Poupon, 2001; Zhu et al., 2001). Pancreatic adenocarci- interference. Using a nude mouse orthotopic xenograft noma is characterized by aggressive invasion and early model, we assessed the effect of this treatment on in vivo metastasis, such that 90% of patients have surgically metastatic ability. Anoikis resistance was associated with unresectable advanced disease at the time of diagnosis. increased CEACAM6 expression. CEACAM6-specific Even patients without overtly advanced disease often short interfering ribonucleic acid (siRNA), but not control subsequently develop metastases and tumor recurrence siRNA, increased susceptibility to caspase-mediated despite apparently curative resection (Farrell et al., anoikis, an effect abrogated by Z-VAD-fmk, and 1997). Anoikis resistance represents a potential target decreased Akt phosphorylation (Ser-473) under ancho- for novel antimetastatic strategies directed at preventing rage-independent conditions. CEACAM6 gene silencing or slowingprogression of this disease. reversed the acquired anoikis resistance of MiaAR and Carcinoembryonic antigen (CEA) and carcinoem- inhibited its in vivo metastatic ability. CEACAM6 bryonic antigen-related cell adhesion molecules (CEA– warrants further investigation as a novel therapeutic CAM), members of the immunoglobulin superfamily target for the treatment of pancreatic adenocarcinoma. (Thompson et al., 1991), function as intercellular Oncogene (2004) 23, 465–473. doi:10.1038/sj.onc.1207036 adhesion molecules (Kuroki et al., 2001). The 90 kDa glycosylphosphatidylinositol (GPI)-linked subtype Keywords: anoikis; pancreatic cancer; adenocarcinoma; CEACAM6, formerly known as nonspecific crossreact- CEACAM6; RNA interference; metastasis ingantigen90 or CD66c (Beauchemin et al., 1999), is found principally on neutrophils and some epithelia (Scholzel et al., 2000). Once thought merely to represent a ‘differentiation marker’, evidence is accumulatingthat CEACAM6, in particular, has functional importance in Introduction tumorigenesis, including disruption of tissue architec- ture, cell polarity, differentiation and anoikis (Ilantzis Epithelial and endothelial cells generally require cell– et al., 2002). Deregulated overexpression of CEACAM6 cell or cell–matrix contact to survive and proliferate. has been reported in numerous human cancers (Hase- The normal cellular response to inadequate or inap- gawa et al., 1993; Kodera et al., 1993; Scholzel et al., propriate intercellular or matrix attachment is a subset 2000) and may promote anoikis resistance (Ordonez of apoptosis termed anoikis. While anoikis is thought to et al., 2000; Ilantzis et al., 2002). Owingto these features of CEACAM6, we tested the *Correspondence: EE Whang; E-mail: [email protected] hypothesis that inhibition of its expression by short Received 29 May 2003; revised 21 July 2003; accepted 23 July 2003 interferingribonucleic acid (siRNA) would impair CEACAM6 and pancreatic adenocarcinoma anoikis MS Duxbury et al 466 anoikis resistance in pancreatic ductal adenocarcinoma was 1.2% compared to 14.1% for MiaPaCa2 (Po0.01. and determined the roles of Akt and caspases as Table 1). This increased anoikis resistance did not mediators of this process. We report for the first time equate to global resistance to apoptosis as MiaAR that suppression of CEACAM6 expression by siRNA remained sensitive to apoptosis induced by 100 nm enhances pancreatic adenocarcinoma cell anoikis, staurosporine. The staurosporine-induced apoptosis increases caspase activation in response to anchorage- fraction was 48% in MiaPaCa2 and 46% in MiaAR. independent conditions, impairs acquired anoikis resistance in a selected anoikis-resistant subline and inhibits metastasis in vivo. Anoikis resistance correlates with in vivo metastatic ability In vivo metastatic ability was assessed in a nude mouse xenograft model. At necropsy 4 weeks following Results orthotopic implantation of 1 Â 106 cells of each adeno- carcinoma cell line, liver metastases were counted and Pancreatic cancer cells display variable anoikis resistance confirmed histologically. The in vivo liver metastatic Anoikis was induced by poly-2-hydroxyethylmethacry- ability of the untreated cell lines corresponded to late (polyHEMA) culture, a technique, originally relative anoikis resistance (Table 2). Mice implanted described by Folkman and Moscona (1978), which has with Capan2 did not develop metastases although been widely used to induce this subset of apoptosis by primary tumor growth was evident. In a separate deprivingcells of contact with substratum (Raz and Ben experiment, there was no evidence of Capan2 metastasis, Ze’ev, 1983; Kim et al., 1999). We observed considerable even 12 weeks followingimplantation. The ability of variation in the susceptibility of pancreatic adenocarci- MiaAR to form liver metastases was significantly greater noma cells to anoikis. The anoikis fraction (ratio of than parental MiaPaCa2, from which it was derived. Of apoptotic cells to total cells), quantified by flow the six mice implanted with MiaAR, all developed cytometric analysis of YO-PRO-1 and propidium iodide metastases by 4 weeks compared to 67% of mice stainingfollowing16 h polyHEMA culture, was greatest implanted with MiaPaCa2 (Po0.05). The median in the well-differentiated cell line Capan2 (anoikis number of metastases was greater in the MiaAR group fraction 27%). Resistance to anoikis was significantly (median 6 versus 2 in MiaPaCa2, Po0.05. Table 2). greater in the poorly differentiated cell lines PANC1 and MiaPaCa2 (anoikis fraction 15 and 14%, respectively, CEACAM6 suppression by siRNA suppresses in vitro Po0.01. Table 1). anoikis resistance and in vivo metastatic ability After eight selecting passages in which nonadherent MiaPaCa2 cells were serially subcultured, MiaAR, a cell Cell lines with greater anoikis resistance and metastatic line capable of proliferatingin suspension, was estab- ability demonstrated higher levels of CEACAM6 lished. The stability of this phenotype was confirmed transcript (Figure 1) and protein expression (Figure 2). after five passages under standard culture conditions. Furthermore, CEACAM6 was overexpressed at mRNA Parental MiaPaCa2 cells are adherent to culture vessel and protein levels in the anoikis-resistant cell line MiaAR surfaces, have spindle-like morphology and grow in a compared to the parental MiaPaCa2 line. Suppression discrete scattered fashion. In contrast, the majority of of CEACAM6 by specific siRNA (siCEACAM6) was MiaAR cells remain in suspension although, when confirmed at the mRNA level by Northern blot adherent to culture flask plastic, MiaAR cells have (Figure 3a), and at the protein level by Western blot rounder morphology and grow in clumped colonies. analysis 48 h post-treatment (Figure 3b). Control MiaAR showed marked anoikis resistance compared to siRNA did not affect CEACAM6 expression. Suppres- parental MiaPaCa2. Followingculture in polyHEMA- sion of CEACAM6 expression by siRNA increased coated wells for 16 h, the anoikis fraction of MiaAR cells anoikis in response to 16 h polyHEMA culture in all cell Table 1 CEACAM6 gene silencing promotes anoikis and reverses acquired anoikis resistance Cell line Anoikis fraction (%) PolyHEMA Control siRNA siCEACAM6 siCEACAM6+Z-VAD-fmk MiaPaCa2 14.170.5 17.070.5 23.471.3* 3.071.0*** MiaAR