Oncogene (2009) 28, 3960–3970 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE Impaired PTPN13 phosphatase activity in spontaneous or HPV-induced squamous cell carcinomas potentiates oncogene signaling through the MAP kinase pathway AC Hoover1, GL Strand2, PN Nowicki3, ME Anderson1, PD Vermeer4, AJ Klingelhutz5, AD Bossler2, JV Pottala4, WJAJ Hendriks6 and JH Lee4,7 1Department of Otolaryngology—Head and Neck Surgery, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA, USA; 2Department of Pathology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA, USA; 3Gynecologic Oncology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA, USA; 4Cancer Biology Research Center, Sanford Research/USD, Sioux Falls, SD, USA; 5Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA, USA; 6Department of Cell Biology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands and 7Sanford ENT-Head and Neck Surgery, Sanford Health, Sioux Falls, SD, USA Human papillomaviruses (HPVs) are a causative factor in frequently target key cellular pathways that are also over 90% of cervical and 25% of head and neck squamous altered in non-viral cancers. Because viral genes alter cell carcinomas (HNSCCs). The C terminus of the high- these pathways in a mechanistically consistent manner, risk HPV 16 E6 oncoprotein physically associates with studies of their function often serve as a starting point to and degrades a non-receptor protein tyrosine phosphatase understanding non-viral mechanisms of transformation. (PTPN13), and PTPN13 loss synergizes with H-RasV12 or In most viral cancers, synergistic cellular changes must ErbB2 for invasive growth in vivo. Oral keratinocytes that occur for malignant progression to occur. Therefore, it have lost PTPN13 and express H-RasV12 or ErbB2 show is important to study viral gene function in the context enhanced Ras/RAF/MEK/Erk signaling. In co-transfec- of these cellular changes. The following study examines tion studies, wild-type PTPN13 inhibited Ras/RAF/ a synergy between human papillomaviruses (HPV) viral MEK/Erk signaling in HEK 293 cells that overexpress oncogene function and cellular changes that lead to ErbB2, EGFR or H-RasV12, whereas an enzymatically invasion. High-risk HPVs promote cancerous growth inactive PTPN13 did not. Twenty percent of HPV- through overexpression of two multifunctional viral negative HNSCCs had PTPN13 phosphatase mutations oncoproteins, E6 and E7. Their known transforming that did not inhibit Ras/RAF/MEK/Erk signaling. functions include inactivation of pRB by E7 and Inhibition of Ras/RAF/MEK/Erk signaling using MEK degradation of p53 and activation of telomerase by E6 inhibitor U0126 blocked anchorage-independent growth in (Longworth and Laimins 2004). E6 oncoproteins from cells lacking PTPN13. These findings show that PTPN13 HPV subtypes that are high risk for malignant progres- phosphatase activity has a physiologically significant role sion also contain a C-terminal PDZ-binding motif in regulating MAP kinase signaling. (PDZBM), which has a poorly understood yet necessary Oncogene (2009) 28, 3960–3970; doi:10.1038/onc.2009.251; role in malignant transformation. PDZBMs are short published online 7 September 2009 C-terminal amino-acid sequences capable of binding PDZ domain containing proteins (Jelen et al., 2003). Keywords: human papilloma virus; HPV E6 oncogene; We have previously investigated the transforming PTPN13; MAP Kinase; ErbB2 effects of the E6 PDZBM of HPV type 16 in HPV- related head and neck squamous cell cancers (HNSCCs) (Spanos et al., 2008b) and cervical cancer (PN Nowicki Introduction et al., unpublished data) and have shown that it physically associates with and induces loss of PTPN13, Malignant transformation often occurs through ran- a non-receptor protein tyrosine phosphatase that con- dom, accumulated genetic changes resulting in char- tains five PDZ domains. In addition, HPV 16 E6 or acteristic features shared by nearly all cancers (Hanahan short hairpin RNA (shRNA) mediated PTPN13 loss V12 and Weinberg 2000). It is estimated that viral gene synergizes with H-Ras for invasive growth in vitro and expression has a role in 20% of cancers. Viral genes in vivo models of HNSCC (Spanos et al., 2008a, 2008b). Besides our data, PTPN13 has been reported as a putative tumor suppressor in a wide range of epithelial Correspondence: JH Lee, Department of Otolaryngology, Sanford cancers (including breast, colon and hepatocellular HPV Research Institute, 1310 22nd Street, Sioux Falls, SD 57105, (Wang et al., 2004; Yeh et al., 2006; Ying et al., 2006)). USA. E-mail: [email protected] Analysis of synergistic changes associated with PTPN13 Received 2 December 2008; revised 6 July 2009; accepted 11 July 2009; loss in colon cancers showed that a majority had published online 7 September 2009 mutations in the MAP kinase pathway (Wang et al., 2004). Oncogene signaling through the MAP kinase pathway AC Hoover et al 3961 Although some reports show significant association V660E expression construct. E6D146À151 is a deletion between Ras mutations and HPV in cervical cancers mutant of HPV16, which degrades p53 but lacks a (Lee et al., 1996; Landro et al., 2008), direct activating PDZBM and does not induce the loss of PTPN13 Ras mutations (like H-RasV12) are less common in (Spanos et al., 2008b). The shPTPN13 cells lack HNSCCs (Yarbrough et al., 1994; Hardisson 2003; Lu PTPN13 due to an shRNA mechanism (Spanos et al., et al., 2006). Ras pathway stimulation may alternatively 2008b). Through western blot, we confirmed that be achieved in HNSCCs by overexpression of mem- retroviral transduction with ErbB2 V660E increases brane-bound growth factor receptors, most notably the total and phoshorylated ErbB2 as compared with the ErbB family of receptor tyrosine kinases. The four parental cells (Figure 1a). Previously described PTPN13 members of this family (ErbB1–4) are commonly levels (Spanos et al., 2008b) were not altered by the overexpressed in HNSCCs and are associated with addition of ErbB2 V660E (not shown). activation of several major cancer-associated signaling To determine whether HPV 16 E6 PDZBM-mediated cascades, including signal transducers and activators degradation of PTPN13 is required for invasive growth of transcription (STAT’s), Ras/RAF/MEK/Erk (MAP in synergy with ErbB2, we injected 1 Â 106 cells from Kinase) and PI3 Kinase/AKT (Ford and Grandis each ErbB2 V660E-expressing cell line subcutaneously 2003). ErbB2 specifically is overexpressed in up to into the right hind legs of C57BL/6 mice (5 mice per 47% of HNSCCs (Cavalot et al., 2007), and when group). Weekly caliper measurements were used to combined with the expression of E6/E7 causes invasive calculate average mouse leg circumferences and estimate growth in primary oral keratinocytes, although the tumor growth rates over time (Figure 1b). All mice mechanism of HPV/ErbB2 synergy and the contribution receiving MTE HPV 16 E6/ErbB2 V660E or MTE of the E6 PDZBM were not explored (Al Moustafa shPTPN13/ErbB2 V660E cells formed tumors and met et al., 2004). criteria for killing within 50 and 30 days, respectively, Therefore, we have investigated whether the common but no mice receiving MTE HPV 16 E6D146À151/ErbB2 HNSCC oncogene, ErbB2, synergizes with HPV 16 E6- V660E cells showed signs of tumor formation for more induced PTPN13 loss to result in invasive growth than 80 days (Figure 1c). Representative mouse legs in vivo. To understand how PTPN13 loss alters cell from each group are shown at 2 weeks post-injection signaling promoting invasion, we investigated the (Figure 1d). MTE cells expressing both HPV 16 E6 and phosphorylation status of the relevant effector pathway E7, and ErbB2 V660E formed tumors at the similar signaling components in the presence or absence of rate as HPV 16 E6/ErbB2 V660E (data not shown). functional PTPN13. We describe a mechanism of Thus, oncogenic forms of ErbB2 (Figure 1) and H-Ras PTPN13’s phosphatase: the regulation MAP Kinase (Spanos et al., 2008b) synergize with HPV 16 E6 in a cascade signaling. Furthermore, we provide evidence similar PDZBM-dependent manner, suggesting that that PTPN13 loss of function may have a crucial role in growth factor receptor-mediated Ras activation may potentiating MAP Kinase cascade signaling down- be important to allow invasive growth in HPV-related stream of multiple different Ras-activating oncogenes HNSCCs. found in both HPV-positive and -negative epithelial cancers. PTPN13 loss correlates with increased Erk phosphorylation Our in vivo findings show that the expression of ErbB2 Results V660E or H-RasV12 is tumorigenic in oropharyngeal keratinocytes only if PTPN13 levels are decreased in ErbB2 synergizes with PTPN13 loss allowing invasive parallel, suggesting a potential role for PTPN13 in the growth in vivo regulation of cancer signaling pathways affected in Previously, we have shown that H-Rasv12 expression common by both ErbB2 and its downstream effector, synergizes with HPV 16 E6 or shRNA-mediated Ras. To assess what common signaling pathways are PTPN13 loss to allow invasive growth (Spanos et al., altered by the loss of PTPN13 and Ras/ErbB2 expres- 2008b). Here, we sought to determine if ErbB2, a sion, we examined phosphorylation levels of Erk and v12 common HNSCC oncogene that activates Ras, would AKT. MTE HPV 16 E6D146À151/H-Ras , MTE HPV also cause invasive growth when PTPN13 is absent. In 16 E6/H-Rasv12 and MTE shPTPN13/ H-Rasv12 cells human cancers, ErbB2 overexpression is oncogenic, but were grown to confluency, serum-starved for 24 h, and in rodent cancer models activating mutations are protein lysates were collected for immunoblot. Although required for efficient tumor formation (Moasser, we observed variation in phospho-AKT levels among 2007). We therefore cloned mouse ErbB2 cDNA into a the MTE cell lines, we found no correlation between retroviral vector and performed site-directed mutagen- phospho-AKT levels, in vivo invasive growth potential esis to introduce a transmembrane region point muta- and the presence/absence of PTPN13.