Characterization of STAT3 expression, signaling and inhibition in feline oral squamous cell carcinoma

BMC Veterinary Research

Cheryl A. London¹  William C. Kisseberth²  Chris Premanandan¹  Thomas J. Rosol¹  Misty D. Bear²  Megan E. Brown² 

[1] Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA;Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA

关键词: Feline;    Oral squamous cell carcinoma;    STAT3;   

Others  :  1224346
DOI  :  10.1186/s12917-015-0505-7

 received in 2015-03-19, accepted in 2015-07-23,  发布年份 2015

【 摘 要 】

Background

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in tumor development by regulating signaling pathways involved in cell proliferation, survival, metastasis and angiogenesis. STAT3 is activated in many cancers, including head and neck squamous cell carcinoma (HNSCC) in people. Feline oral squamous cell carcinoma (OSCC) is similar to advanced or recurrent HNSCC as it is poorly responsive to traditional therapies and carries a poor long-term prognosis. The purpose of this study was to characterize expression and activation of STAT3 in feline OSCC cell lines and tumor samples and to investigate the biologic activity of a novel, allosteric STAT3 inhibitor, LLL12, in feline OSCC cell lines.

Results

We evaluated 3 feline OSCC cell lines and one of these (SCCF2) exhibited high levels of constitutive STAT3 phosphorylation and high sensitivity to LLL12 treatment. Exposure of SCCF2 cells to LLL12 resulted in decreased expression of pSTAT3 and total STAT3, apoptosis as assessed by caspase 3/7 activation, inhibition of colony formation and reduced expression of the STAT3 transcriptional target survivin. In contrast, the STAT3 transcriptional targets VEGF and MCL-1 increased after LLL12 treatment. This was, in part, likely due to LLL12 mediated upregulation of HIF-1α, which is known to drive VEGF and MCL-1 expression. The OSCC cell lines with low basal STAT3 phosphorylation did not exhibit these effects, suggesting that STAT3 inhibition was responsible for the observed results. Lastly, immunohistochemistry for pSTAT3 was performed using a feline OSCC tissue microarray, demonstrating expression in 48 % of samples tested.

Conclusions

These data demonstrate that LLL12 has biologic activity against a feline OSCC cell line expressing pSTAT3 and that STAT3 represents a target for therapeutic intervention in this disease. However, given the up-regulation of several STAT3 transcriptional targets following treatment, further investigation of STAT3 and its related signaling pathways in OSCC is warranted.

【 授权许可】

   
2015 Brown et al.

【 图 表 】

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 7.

Fig. 8.

【 参考文献 】

• [1]Bowman T, Garcia R, Turkson J, Jove R. STATs in oncogenesis. Oncogene. 2000; 19:2474-2488.
• [2]Turkson J, Bowman T, Garcia R, Caldenhoven E, De Groot RP, Jove R. Stat3 activation by Src induces specific gene regulation and is required for cell transformation. Mol Cell Biol. 1998; 18:2545-2552.
• [3]Turkson J, Jove R. STAT proteins: novel molecular targets for cancer drug discovery. Oncogene. 2000; 19:6613-6626.
• [4]Garcia R, Bowman TL, Niu G, Yu H, Minton S, Muro-Cacho CA et al.. Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells. Oncogene. 2001; 20:2499-2513.
• [5]Silva CM. Role of STATs as downstream signal transducers in Src family kinase-mediated tumorigenesis. Oncogene. 2004; 23:8017-8023.
• [6]Huang S. Regulation of metastases by signal transducer and activator of transcription 3 signaling pathway: clinical implications. Clin Cancer Res. 2007; 13:1362-1366.
• [7]Leeman RJ, Lui VWY, Grandis JR. STAT3 as a therapeutic target in head and neck cancer. Expert Opin Biol Ther. 2006; 6:231-241.
• [8]Komazaki T, Nagai H, Emi M, Terada Y, Yabe A, Jin E et al.. Hypermethylation-associated inactivation of the SOCS-1 gene, a JAK/STAT inhibitor, in human pancreatic cancers. Jpn J Clin Oncol. 2004; 34:191-194.
• [9]Sen M, Thomas SM, Kim S, Yeh JI, Ferris RL, Johnson JT et al.. First-in-human trial of a STAT3 decoy oligonucleotide in head and neck tumors: implications for cancer therapy. Cancer Discov. 2012; 2:694-705.
• [10]Bendell JC, Hong DS, Burris HA, Naing A, Jones SF, Falchook G et al.. Phase 1, open-label, dose-escalation, and pharmacokinetic study of STAT3 inhibitor OPB-31121 in subjects with advanced solid tumors. Cancer Chemother Pharmacol. 2014; 74:125-130.
• [11]Grandis JR, Drenning SD, Chakraborty A, Zhou M-Y, Zeng Q, Pitt AS et al.. Requirement of Stat3 but not Stat1 activation for epidermal growth factor receptor-mediated cell growth In vitro. J Clin Invest. 1998; 102:1385.
• [12]Grandis JR, Drenning SD, Zeng Q, Watkins SC, Melhem MF, Endo S et al.. Constitutive activation of Stat3 signaling abrogates apoptosis in squamous cell carcinogenesis in vivo. Proc Natl Acad Sci U S A. 2000; 97:4227-4232.
• [13]Masuda M, Wakasaki T, Suzui M, Toh S, Joe AK, Weinstein IB. Stat3 orchestrates tumor development and progression: the Achilles’ heel of head and neck cancers? Curr Cancer Drug Targets. 2010; 10:117-126.
• [14]Macha MA, Matta A, Kaur J, Chauhan SS, Thakar A, Shukla NK et al.. Prognostic significance of nuclear pSTAT3 in oral cancer. Head Neck. 2011; 33:482-489.
• [15]Stebbins KE, Morse CC, Goldschmidt MH. Feline oral neoplasia: a ten-year survey. Vet Pathol. 1989; 26:121-128.
• [16]Postorino Reeves NC, Turrel JM, Withrow SJ. Oral squamous cell carcinoma in the cat. J Am Anim Hosp Assoc. 1993; 29:1-4.
• [17]Hutson CA, Willauer CC, Walder EJ, Stone JL, Klein MK. Treatment of mandibular squamous cell carcinoma in cats by use of mandibulectomy and radiotherapy: seven cases (1987–1989). J Am Vet Med Assoc. 1992; 201:777-781.
• [18]Northrup NC, Selting KA, Rassnick KM, Kristal O, O’Brien MG, Dank G et al.. Outcomes of cats with oral tumors treated with mandibulectomy: 42 cases. J Am Anim Hosp Assoc. 2006; 42:350-360.
• [19]Fidel JL, Sellon RK, Houston RK, Wheeler BA. A nine-day accelerated radiation protocol for feline squamous cell carcinoma. Vet Radiol Ultrasound. 2007; 48:482-485.
• [20]Sabhlok A, Ayl R. Palliative radiation therapy outcomes for cats with oral squamous cell carcinoma (1999–2005). Vet Radiol Ultrasound. 2014; 0:1-6.
• [21]Fox LE, Rosenthal RC, King RR, Levine PB, Vail DM, Helfand SC et al.. Use of cis-bis-neodecanoato-trans-R, R-1,2-diaminocyclohexane platinum (II), a liposomal cisplatin analogue, in cats with oral squamous cell carcinoma. Am J Vet Res. 2000; 61:791-795.
• [22]Fidel J, Lyons J, Tripp C, Houston R, Wheeler B, Ruiz A. Treatment of oral squamous cell carcinoma with accelerated radiation therapy and concomitant carboplatin in cats. J Vet Intern Med. 2011; 25:504-510.
• [23]LeBlanc AK, LaDue TA, Turrel JM, Klein MK. Unexpected toxicity following use of gemcitabine as a radiosensitizer in head and neck carcinomas: a veterinary radiation therapy oncology group pilot study. Vet Radiol Ultrasound. 2004; 45:466-470.
• [24]Lin L, Hutzen B, Li P-K, Ball S, Zuo M, DeAngelis S et al.. A novel small molecule, LLL12, inhibits STAT3 phosphorylation and activities and exhibits potent growth-suppressive activity in human cancer cells. Neoplasia. 2010; 12:39-51.
• [25]Onimoe G-I, Liu A, Lin L, Wei C-C, Schwartz EB, Bhasin D et al.. Small molecules, LLL12 and FLLL32, inhibit STAT3 and exhibit potent growth suppressive activity in osteosarcoma cells and tumor growth in mice. Invest New Drugs. 2011; 30:916-926.
• [26]Bid HK, Oswald D, Li C, London CA, Lin J, Houghton PJ. Anti-angiogenic activity of a small molecule STAT3 inhibitor LLL12. PLoS One. 2012; 7:e35513.
• [27]Tannehill-Gregg S, Kergosien E, Rosol TJ. Feline head and neck squamous cell carcinoma cell line: characterization, production of parathyroid hormone-related protein, and regulation by transforming growth factor-beta. In Vitro Cell Dev Biol Anim. 2001; 37:676-683.
• [28]Martin CK, Dirksen WP, Shu ST, Werbeck JL, Thudi NK, Yamaguchi M et al.. Characterization of bone resorption in novel in vitro and in vivo models of oral squamous cell carcinoma. Oral Oncol. 2012; 48:491-499.
• [29]Buch K, Peters T, Nawroth T, Sänger M, Schmidberger H, Langguth P. Determination of cell survival after irradiation via clonogenic assay versus multiple MTT Assay - a comparative study. Radiat Oncol. 2012; 7:1. BioMed Central Full Text
• [30]Fossey SL, Liao AT, McCleese JK, Bear MD, Lin J, Li P-K et al.. Characterization of STAT3 activation and expression in canine and human osteosarcoma. BMC Cancer. 2009; 9:81. BioMed Central Full Text
• [31]Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nat Protoc. 2008; 3:1101-1108.
• [32]Martin CK, Tannehill-Gregg SH, Wolfe TD, Rosol TJ. Bone-invasive oral squamous cell carcinoma in cats: pathology and expression of parathyroid hormone-related protein. Vet Pathol. 2011; 48:302-312.
• [33]Ulane CM, Rodriguez JJ, Parisien JP, Horvath CM. STAT3 ubiquitylation and degradation by mumps virus suppress cytokine and oncogene signaling. J Virol. 2003; 77:6385-6393.
• [34]Safhi MMA. Priming of STAT1 and STAT3 for cytokine-triggered degradation by the proteasome upon A2Aadenosine Receptor (A2AAR) expression. Mol Pharmacol. 2008; 77:968-78.
• [35]Ichiba M, Nakajima K, Yamanaka Y, Kiuchi N, Hirano T. Autoregulation of the Stat3 gene through cooperation with a cAMP-responsive element-binding protein. J Biol Chem. 1998; 273:6132-6138.
• [36]Tannehill-Gregg SH, Levine AL, Rosol TJ. Feline head and neck squamous cell carcinoma: a natural model for the human disease and development of a mouse model. Vet Comp Oncol. 2006; 4:84-97.
• [37]Wypij JM. A naturally occurring feline model of head and neck squamous cell carcinoma. Pathol Res Int. 2013; 2013:1-7.
• [38]Munday JS, French AF, Gibson IR, Knight CG. The presence of p16CDKN2A protein immunostaining within feline nasal planum squamous cell carcinomas is associated with an increased survival time and the presence of papillomaviral DNA. Vet Pathol. 2013; 50:269-273.
• [39]Munday JS, Gibson I, French AF. Papillomaviral DNA and increased p16CDKN2A protein are frequently present within feline cutaneous squamous cell carcinomas in ultraviolet-protected skin. Vet Dermatol. 2011; 22:360-366.
• [40]Munday JS, Knight CG, French AF. Research in veterinary science. Res Vet Sci. 2011; 90:280-283.
• [41]Bertone ER, Snyder LA, Moore AS. Environmental and lifestyle risk factors for oral squamous cell carcinoma in domestic cats. J Vet Intern Med. 2003; 17:557-562.
• [42]Snyder LA, Bertone ER, Jakowski RM, Dooner MS, Jennings-Ritchie J, Moore AS. p53 expression and environmental tobacco smoke exposure in feline oral squamous cell carcinoma. Vet Pathol. 2004; 41:209-214.
• [43]Looper JS, Malarkey DE, Ruslander D, Proulx D, Thrall DE. Epidermal growth factor receptor expression in feline oral squamous cell carcinomas. Vet Comp Oncol. 2006; 4:33-40.
• [44]Bergkvist GT, Argyle DJ, Morrison L, MacIntyre N, Hayes A, Yool DA. Expression of epidermal growth factor receptor (EGFR) and Ki67 in feline oral squamous cell carcinomas (FOSCC). Vet Comp Oncol. 2010; 9:106-117.
• [45]Bergkvist GT, Argyle DJ, Pang LY, Muirhead R, Yool DA. Studies on the inhibition of feline EGFR in squamous cell carcinoma: enhancement of radiosensitivity and rescue of resistance to small molecule inhibitors. Cancer Biol Ther. 2011; 11:927-937.
• [46]Masuda M, Suzui M, Yasumatu R, Nakashima T, Kuratomi Y, Azuma K et al.. Constitutive activation of signal transducers and activators of transcription 3 correlates with cyclin D1 overexpression and may provide a novel prognostic marker in head and neck squamous cell carcinoma. Cancer Res. 2002; 62:3351-3355.
• [47]Shah NG, Trivedi TI, Tankshali RA, Goswami JA, Jetly DH, Kobawala TP et al.. Stat3 expression in oral squamous cell carcinoma: association with clinicopathological parameters and survival. Int J Biol Markers. 2005; 21:175-183.
• [48]Pectasides E, Egloff AM, Sasaki C, Kountourakis P, Burtness B, Fountzilas G et al.. Nuclear localization of signal transducer and activator of transcription 3 in head and neck squamous cell carcinoma is associated with a better prognosis. Clin Cancer Res. 2010; 16:2427-2434.
• [49]Seethala RR, Gooding WE, Handler PN, Collins B, Zhang Q, Siegfried JM, Grandis JR. Immunohistochemical analysis of phosphotyrosine signal transducer and activator of transcription 3 and epidermal growth factor receptor autocrine signaling pathways in head and neck cancers and metastatic lymph nodes. Clin Cancer Res. 2008; 14:1303-1309.
• [50]Couto JI, Bear MD, Lin J, Pennel M, Kulp SK, Kisseberth WC et al.. Biologic activity of the novel small moleculeSTAT3 inhibitor LLL12 against canineosteosarcoma cell lines. BMC Vet Res. 2012; 8:244. BioMed Central Full Text
• [51]Yue P, Zhang X, Paladino D, Sengupta B, Ahmad S, Holloway RW et al.. Hyperactive EGF receptor, Jaks and Stat3 signaling promote enhanced colony-forming ability, motility and migration of cisplatin-resistant ovarian cancer cells. Oncogene. 2011; 31:2309-2322.
• [52]Reed S, Li H, Li C, Lin J. Celecoxib inhibits STAT3 phosphorylation and suppresses cell migration and colony forming ability in rhabdomyosarcoma cells. Biochem Biophys Res Commun. 2011; 407:450-455.
• [53]Wang X, Goldstein D, Crowe PJ, Yang J-L. Impact of STAT3 inhibition on survival of osteosarcoma cell lines. Anticancer Res. 2014; 34:6537-6545.
• [54]Darnowski JW, Goulette FA, Guan Y-J, Chatterjee D, Yang Z-F, Cousens LP et al.. Stat3 cleavage by caspases impact on full-length Stat3 expression, fragment formation, and transcriptional activity. J Biol Chem. 2006; 281:17707-17717.
• [55]Haghikia A, Hoch M, Stapel B, Hilfiker-Kleiner D. STAT3 regulation of and by microRNAs in development and disease. JAKSTAT. 2014; 1:143-150.
• [56]Klosek SK, Nakashiro K-I, Hara S, Li C, Shintani S, Hamakawa H. Constitutive activation of Stat3 correlates with increased expression of the c-Met/HGF receptor in oral squamous cell carcinoma. Oncol Rep. 2004; 12:293-296.
• [57]Nagpal JK, Mishra R, Das BR. Activation of Stat-3 as one of the early events in tobacco chewing-mediated oral carcinogenesis. Cancer. 2002; 94:2393-2400.
• [58]Yang J, Chatterjee-Kishore M, Staugaitis SM, Nguyen H, Schlessinger K, Levy DE et al.. Novel roles of unphosphorylated STAT3 in oncogenesis and transcriptional regulation. Cancer Res. 2005; 65:939-947.
• [59]Liu L, McBride KM, Reich NC. STAT3 nuclear import is independent of tyrosine phosphorylation and mediated by importin-alpha3. Proc Natl Acad Sci U S A. 2005; 102:8150-8155.
• [60]Leong PL, Andrews GA, Johnson DE, Dyer KF, Xi S, Mai JC et al.. Targeted inhibition of Stat3 with a decoy oligonucleotide abrogates head and neck cancer cell growth. Proc Natl Acad Sci U S A. 2003; 100:4138-4143.
• [61]Lui VWY, Hedberg ML, Li H, Vangara BS, Pendleton K, Zeng Y et al.. Frequent mutation of the PI3K pathway in head and neck cancer defines predictive biomarkers. Cancer Discov. 2013; 3:761-769.
• [62]Rebucci M, Peixoto P, Dewitte A, Wattez N, De Nuncques MA, Rezvoy N, Vautravers-Dewas C et al.. Mechanisms underlying resistance to cetuximab in the HNSCC cell line: role of AKT inhibition in bypassing this resistance. Int J Oncol. 2010; 38:189-200.
• [63]Abell K, Bilancio A, Clarkson RWE, Tiffen PG, Altaparmakov AI, Burdon TG et al.. Stat3-induced apoptosis requires a molecular switch in PI(3)K subunit composition. Nat Cell Biol. 2005; 7:392-398.
• [64]Jung JE, Kim HS, Lee CS, Shin Y-J, Kim Y-N, Kang G-H et al.. STAT3 inhibits the degradation of HIF-1α by pVHL-mediated ubiquitination. Exp Mol Med. 2008; 40:479.
• [65]Jung JE. STAT3 is a potential modulator of HIF-1-mediated VEGF expression in human renal carcinoma cells. FASEB J. 2005; 10:1296-8.
• [66]Niu G, Briggs J, Deng J, Ma Y, Lee H, Kortylewski M et al.. Signal transducer and activator of transcription 3 is required for hypoxia-inducible factor-1 RNA expression in both tumor cells and tumor-associated myeloid cells. Mol Cancer Res. 2008; 6:1099-1105.
• [67]Ganji PN, Park W, Wen J, Mahaseth H, Landry J, Farris AB et al.. Antiangiogenic effects of ganetespib in colorectal cancer mediated through inhibition of HIF-1α and STAT-3. Angiogenesis. 2013; 16:903-917.
• [68]Carpenter R, Lo H-W. STAT3 target genes relevant to human cancers. Cancers. 2014; 6:897-925.
• [69]Couto JP, Daly L, Almeida A, Knauf JA, Fagin JA, Sobrinho-Simões M et al.. STAT3 negatively regulates thyroid tumorigenesis. Proc Natl Acad Sci U S A. 2012; 109:E2361-E2370.