Letters to the Editor 207

Department of Dermatology,Affiliated Union Hospital, Tongji Medical College,Huazhong University of Science and Technology,

Wuhan 430022, China

Li LiXue Wen

Guan-Xin ShenDepartment of Immunology, Tongji Medical College,

Huazhong University of Science and Technology,Wuhan, China

Ya-Ting Tu*Department of Dermatology,

Affiliated Union Hospital,

KEYWORDSCadherin; Fat2; Migration; Actin; Filopodia

Tongji Medical College,Huazhong University of Science and Technology,

Jiefang Road 1277, Wuhan 430022, China

*Corresponding author. Tel.: +86 27 85726157E-mail address: yatingtu@yahoo.com.cn(Ya-Ting Tu)

1These authors contributed equally to this work.

28 December 2007

doi:10.1016/j.jdermsci.2008.04.012

CORRESPONDENCE

Knockdown of Fat2 by siRNA inhibits themigration of human squamous carcinomacells

To the Editor,

Human Fat2 is one of the huge Fat cadherin familyproteins that contain 34 cadherin repeats [1]. Fat2 isexpressed in thin parallel fibers of cerebellar gran-ule cells [2], epidermal keratinocytes and squamouscell carcinoma (SCC) [3], but the function of Fat2 isnot well understood. In this study, we found thatFat2 functions in the migration of epidermal cells.

To investigate the function of Fat2 in human cuta-neous SCC cell line HSC-1 cells, we examined theeffect of knockdown of Fat2 by the following smallinterfering RNAs (siRNA): siRNA-1: 50-GGGCUUUCAC-CUACCACCUUCCAAA-30 and 50-UUUGGAAGGUGGU-AGGUGAAAGCCC-30; siRNA-2: 50-CAUCUGCAAGGU-GACUGCCACAGAU-30 and 50-AUCUGUGGCAGUCAC-CUUGCAGAUG-30; siRNA-3: 50-GGUUGUCAAUGUGU-CUGAUAUCAAU-30 and 50-AUUGAUAUCAGACACA-UUGACAACC-30 (Stealth Select siRNA; Invitrogen,Carlsbad, CA). Nonspecific siRNA, which was nothomologous to any human gene, was used as a nega-tive control (Invitrogen). The siRNA transfection effi-ciency was >90% using BLOCK-iT Alexa Fluor RedFluorescent Oligo (Invitrogen). Transfection of HSC-1 cells with 10 nM Fat2-specific siRNA or nonspecific

control siRNA inRNAiMAX (Invitrogen)wasperformedfor 48 h. Then immunoblotting using the generatedanti-human Fat2 polyclonal antibody (1:100 dilution)was performed as previously described [3]. Transfec-tion with Fat2-specific siRNA, in comparison withnonspecific control siRNA, resulted in a strongdecrease in the level of Fat2 protein (Fig. 1a). Knock-down of Fat2 was also confirmed at 72 h after trans-fection (data not shown). E-cadherin was alsodetected using mouse anti-human E-cadherin mono-clonal antibody HECD-1 (a kind gifted from Dr. M.Takeichi). E-cadherin expression was not decreasedby any of the siRNAs used. All signalswere normalizedby the expression of actin using mouse anti-humanbeta actin (AC-15) monoclonal antibody (Abcam,Cambridge, UK).

To analyze the effect of Fat2 knockdown on HSC-1cells migration, we performed three different migra-tion assays. First, in a transwell assay, cell migrationwas evaluatedwith a CHEMICONQCMMigration Assaykit (CHEMICON, Temecula, CA), based on the Boydenchamber principle. Cells were added to the upperchamberwithan 8-mmpore size polycarbonatemem-brane, and then culture medium containing 10% FBSwas added to the lower chamber. After 24 h, chemo-tactic cell migration was decreased by all threesiRNAs in comparison with the control (Fig. 1b). Sec-ond, we then performed a phagocytic gold motilityassay, which evaluates random and non-vectorialmotility called chemokinesis. Cells were seeded onthe cover slips which were coated with colloidal goldparticles as described [4] and cultured for 24 h.Photomicrographs of the migrated cells wereobtained and the areas where the cells had removed

208 Letters to the Editor

Fig. 1 Knockdown of Fat2 expression inhibits HSC-1 cells migration. (a) Western blots with antibodies against Fat2, E-cadherin and actin. HSC-1 cells were transfected with each siRNA (lane 1: nonspecific control siRNA, lane 2: RNAi-1, lane3: RNAi-2, and lane 4: RNAi-3). Molecular weight markers are indicated on the right side. (b) Transwell assay. Results arethe means � S.D. of three independent wells. (c) Cell migration on a colloidal gold particle-coated plate. The area wheresingle cells had migrated was measured. Results are the means � S.D. of 30 independent cells. In photographs, clearedareas resulted from the phagocytosis of migrating cells. (d) Cell migration after wounding of monolayer. Photographsshow wounded monolayer at the indicated time point. Dashed lines indicate the wound edge. The area of the woundedmonolayer covered by cells was measured. Results are the means � S.D. of three independent experiments performed inthree fields. (e) Immunofluorescence microscopy in migrating cells (d). Arrows show the formation of actin-rich filopodia.In the right panel, the same slides were observed at higher magnification. Scale bar: 20 mm (b, c, d, and e) HSC-1 cellswere transfected with the indicated siRNA (RNAi; RNAi-1). *p < 0.01 vs. control by Student’s t-test.

Letters to the Editor 209

Fig. 2 Influence of knockdown of Fat2 on cell proliferation and cell aggregation. (a) WST-1 assay. (b) BrdU incorpora-tion. (a and b) Cells were cultured for the indicated time period after trasnsfection with nonspecific control siRNA(control) or Fat2-specific siRNA (RNAi; RNAi-1). Results are the means � S.D. of quadruplicate samples. There was nosignificant difference according to Student’s t test. (c) Aggregation assay of HSC-1 cells, which were transfected withnonspecific (control) or Fat2-specific siRNA (RNAi; RNAi-1). Cells were treated with trypsin in the presence or absence ofCa2+. The cell suspension in HCMF containing Ca2+ was allowed to aggregate for 20 min at 37 8C on a gyratory shaker.

the colloidal gold particles were measured using thepublic Scion Image Beta 4.02 (Scion Corporation,Frederick, MD). Knockdown of Fat2 also decreasedthe phagocytic cell migration (Fig. 1c). Third, weperformed a scratch-wounded migration assay. Cellswere seeded and grown to confluence in 6-wellplates, and the monolayers were partially denudedusing a 1000-ml plastic pipette tip. Loose cells wereremoved by replacement of the medium. Photomi-crographs were obtained at 20 h after injury using amicroscope and cellular movement was quantitatedby planimetry of the denuded area and converted todistance migrated using the public Scion Image Beta4.02 (Scion Corporation). In scratch-wounded cellmonolayers, the migration of cells treated with spe-cific Fat2 siRNAwas significantlydecreasedcomparedwith thecontrolmigration (49.6%decrease) (Fig. 1d).These results in multiple migration assays indicatethat Fat2 expression is required for the migration ofHSC-1 cells.

Next, we investigated how Fat2 regulates cellmigration. In crawling cells, there are protrusionsat the leading edge which are induced by actinreorganization [5]. That is, actin filaments formslender spikes called filopodia at the leading edgethat form focal adhesions with the substratum,linking the substratum to the cell surface [6]. Wepreviously have reported that Fat2 and actin fila-ments are structurally related, as shown by experi-ments, using an inhibitor of actin polymerization[3]. To investigate the effect of Fat2 knockdown onthe formation of the leading edge in migrating cells,immunostaining of the actin cytoskeleton was per-formed using rhodamine phalloidin (Invitrogen).Knockdown of Fat2 reduced the formation of filo-podia compared with the control. Moreover, theleading edge of Fat2-knockdown cells was flat, incontrast to the control (Fig. 1e). It has beenreported that disruption of filopodia formation leads

to inhibition of cell migration [7,8]. Thus, it seemslikely that the reduction in filopodial formation isthe crucial factor in the suppression of cell migra-tion by Fat2 knockdown. But, the mechanism ofreduction of filopodia by Fat2 knockdown stillremains unclear.

Various functions of cadherins have beenreported [9], such as roles in proliferation [4],cell—cell adhesiveness and differentiation. Weexamined the effect of Fat2 knockdown on cellproliferation and cell aggregation in HSC-1 cells.To assess cell proliferation, we performed theWST-1 cell proliferation assay and the BrdU incor-poration assay using Premix WST-1 Cell ProliferationAssay System (Takara Bio, Shiga, Japan) and CellProliferation Biotrack ELISA System, ver2 (Amer-sham Bioscience, Piscataway, NJ), respectively.Knockdown of Fat2 tended to result in slightly highercell proliferation than that in the control, but thedifference was not significant (Fig. 2a, b). Cadherin-mediated cell aggregation was assayed as described[10]. Briefly, cells were treated with 0.01% trypsin inthe presence of 1 mM CaCl2 at 37 8C for 20 min, andthen washed with Ca2+- and Mg2+-free Herpes-buf-fered (pH 7.4) HBSS (HCMF) to obtain single cellsuspensions. Cells in 0.5 mL of 1% albumin in HCMFwith or without 1 mM CaCl2 were incubated in albu-min-coated 24-well plates on a rotary shaker(80 rpm) at 37 8C for 20 min. In the aggregationassay, Fat2 knockdown cells formed into a largeaggregate in the presence of Ca2+, as did the controlcells (Fig. 2c).

In conclusion, we demonstrated in this study thatexpression of Fat2 plays an important role in themigration of HSC-1 cells. Our new findings on thefunction of Fat2 will provide clues for clarifying themechanism of migration of epidermal cells andnovel therapeutic targets for the invasion of squa-mous cell carcinoma.

210 Letters to the Editor

References

[1] Tanoue T, Takeichi M. New insights into Fat cadherins. J CellSci 2005;118:2347—53.

[2] Nakayama M, Nakajima D, Yoshimura R, Endo Y, Ohara O.MEGF1/fat2 proteins containing extraordinarily large extra-cellular domains are localized to thin parallel fibers of cer-ebellar granule cells. Mol Cell Neurosci 2002;20:563—78.

[3] Matsui S, Utani A, Takahashi K, Mukoyama Y, Miyachi Y,Matsuyoshi N. Human Fat2 is localized at immature adherensjunctions in epidermal cells. J Dermatol Sci 2007;48(3):233—6.

[4] Mukoyama Y, Zhou S, Miyachi Y, Matsuyoshi N. T-cadherinnegatively regulates the proliferation of cutaneous squamouscarcinoma cells. J Invest Dermatol 2005;124:833—8.

[5] Vicente-Manzanares M, Webb DJ, Horwitz AR. Cell migrationat a glance. J Cell Sci 2005;118:4917—9.

[6] Pollard TD, Borisy GG. Cellular motility driven by assemblyand disassembly of actin filaments. Cell 2003;112: 453—65.

[7] Nishita M, Yoo SK, Nomachi A, Kani S, SougawaN, Ohta Y, et al.Filopodia formation mediated by receptor tyrosine kinaseRor2 is required for Wnt5a-induced cell migration. J Cell Biol2006;175:555—62.

[8] Bennett RD, Mauer AS, Strehler EE. Calmodulin-like proteinincreases filopodia-dependent cell motility via up-regulationof myosin-10. J Biol Chem 2007;282:3205—12.

[9] Angst BD, Marcozzi C, Magee AI. The cadherin superfamily:diversity in form and function. J Cell Sci 2001;53:629—41.

[10] Takeichi M. Functional correlation between cell adhesiveproperties and some cell surface proteins. J Cell Biol1977;75:464—74.

Seiya Matsui*Department of Dermatology,Graduate School of Medicine,

Kyoto University, Kyoto, Japan

Drug Discovery Research Laboratories,Maruho Co., Ltd., 92 Awata-cho,

KEYWORDSATF2; STAT3; Squamous cell carcinoma; Bowen’s disease;Basal cell carcinoma

Chudoji, Simogyo-ku,Kyoto 600-8815, Japan

Atsushi UtaniKenzo Takahashi

Department of Dermatology,Graduate School of Medicine,

Kyoto University,Kyoto, Japan

Yohei MukoyamaDrug Discovery Research Laboratories,

Maruho Co., Ltd.,92 Awata-cho, Chudoji, Simogyo-ku,

Kyoto 600-8815, Japan

Yoshiki MiyachiNorihisa Matsuyoshi

Department of Dermatology,Graduate School of Medicine,

Kyoto University,Kyoto, Japan

*Corresponding author at: Drug Discovery ResearchLaboratories, Maruho Co., Ltd.,

92 Awata-cho, Chudoji, Simogyo-ku,Kyoto 600-8815, Japan.

Tel.: +81 75 325 3250; fax: +81 75 325 3471

E-mail address: matsui_ckk@mii.maruho.co.jp(S. Matsui)

29 January 2008

doi:10.1016/j.jdermsci.2008.04.006

LETTER TO THE EDITOR

Overexpression of phosphorylated-ATF2and STAT3 in cutaneous squamous cellcarcinoma, Bowen’s disease and basalcell carcinoma

Squamous cell carcinoma (SCC), Bowen’s disease(BD) and basal cell carcinoma (BCC) are the mostfrequently occurring tumors of the skin, but their

precise molecular pathogenesis remains unclear. Inthe present study, activation of activating transcrip-tion factor-2 (ATF2) and signal transducer and acti-vator of transcription-3 (STAT3) are examined inthese common cutaneous malignancies becauseextracellular signal-regulated kinases (ERK) andAP-1 have been shown to play important roles inthe development of SCC and/or BCC [1—3], andATF2- and STAT3-pathways could be significantlyrelated to these transcription-associated mole-cules. Activation of both ATF2- and STAT3-pathwayshas been suggested in several tumors, such as breastcancer, melanoma or eccrine gland tumors [4—6],but ATF2 activation and the relationship with STAT3