Peptidyl-tRNA hydrolase 2 (PTRH2) regulates integrin-mediated pro-survival and apoptotic signaling. PTRH2 is critical in muscle development and regulates myogenic differentiation. In humans a biallelic mutation in the PTRH2 gene causes infantile-onset multisystem disease with progressive muscle weakness. We report here that the Ptrh2 knockout mouse model recapitulates the progressive congenital muscle pathology observed in patients. Ptrh2 null mice demonstrate multiple degenerating and regenerating muscle fibers, increased central nuclei, elevated creatine kinase activity and endomysial fibrosis. This progressive muscle pathology resembles the muscular dystrophy phenotype in humans and mice lacking the α7 integrin. We demonstrate that in normal muscle Ptrh2 associates in a complex with the α7β1 integrin at the sarcolemma and Ptrh2 expression is decreased in α7 integrin null muscle. Furthermore, Ptrh2 expression is altered in skeletal muscle of classical congenital muscular dystrophy mouse models. Ptrh2 levels were up-regulated in dystrophin deficient mdx muscle, which correlates with the elevated levels of the α7β1 integrin observed in mdx muscle and Duchenne muscular dystrophy patients. Similar to the α7 integrin, Ptrh2 expression was decreased in laminin-α2 dyW null gastrocnemius muscle. Our data establishes a PTRH2 mutation as a novel driver of congenital muscle degeneration and identifies a potential novel target to treat muscle myopathies.
The human papillomavirus (HPV) expresses the viral oncogene E7 that inhibits the retinoblastoma protein (RB1). RB1 mediates contradictory cell growth and cell death pathways via E2F family members. Here, we assessed the extent to which HPV oncogenes caused toxicity as measured by mouse survival and tumor growth. Materials/Methods: iHPV mice contained a LoxP-Stop-LoxP (LSL)-iE6E7 transgene in which conditional E6E7 expression is regulated by Cre recombinase. We constitutively expressed HPV oncogenes by breeding iHPV transgenic mice to CMV-Cre transgenic mice expressing Cre recombinase under a CMV promoter (CMV-HPV mice). We induced HPV oncogene expression in adult mice using RosaHPV mice containing the iHPV transgene and a Rosa-CreER tam transgene expressing a tamoxifenregulated (TAM) regulated Cre recombinase in all tissues. We studied primary oral tumors using triple transgenic KHR mice containing a K14-CreER tam transgene, a LSL-iE6E7 transgene and a LSL-Kras G12D transgene that formed HPV-positive oral tumors after TAM treatment. We assessed the role of E2f1 on survival and oral tumor formation using RosaHPV-E2f1-/mice and KHR-E2f1-/mice, respectively, that contained a homozygous E2f1deletion. HPV oncogene expression and E2f target gene expression was assessed by quantitative RT-PCR. Results: Induction of HPV oncogenes caused embryonic lethality as CMV-HPV double transgenic mice were born at significantly lower frequencies compared to mice carrying single transgenes (P<.0001). Tamoxifen treatment of adult RosaHPV (RosaHPV+TAM) mice caused recombination of the LSL-E6E7 transgenes and HPV oncogene expression in all organs tested. Furthermore, RosaHPV+TAM mice had decreased survival compared to vehicle treated RosaHPV mice (median survival: 50d for RosaHPV+TAM vs not reached for RosaHPV-TAM; P<.0001). Decreased survival in RosaHPV+TAM mice was associated with focal necrosis in hepatocytes and pancreatic tissues and the activation of the E2f target genes. Deletion of E2f1 increased survival of RosaHPV+TAM mice indicating that E2f1 mediated HPV oncogene toxicity (median survival: not reached for RosaHPV-E2f1-/mice vs 49 days for RosaHPV-E2f1 +/mice vs 30 days for RosaHPV-E2f1 +/+ ; P<.0001). Compared to tumors with heterozygous loss of E2f1, KHR tumors with homozygous loss of E2f1 grew faster and had more proliferating tumor cells as measured by Pcna immunohistochemistry (tumor volume at d18: 453.7 mm 3 for KHR-E2f1-/vs 139.7 mm 3 for KHR-E2f1 +/-; PZ.0004). Conclusion: Our results indicate that HPV oncogenes activated the E2f1 pathway to cause toxicity in normal mice and to suppress oral tumor growth. These results suggest that selective modulation of the E2f1 pathway, which is activated in HPV tumors, may facilitate tumor regression.
Human papillomaviruses (HPVs) are etiological agents of many anogenital and oropharyngeal cancers. HPV[+] oropharyngeal squamous cell carcinomas (OPSCCs) typically respond more favorably to current treatment regimens (including radiotherapy combined with cisplatin or cetuximab, an EGFR monoclonal antibody), than do HPV-negative OPSCCs. The discrepancy in patient outcomes has been attributed, in part, to substantially fewer genetic mutations present in HPV[+] cancers. Paradoxically, an increasing body of literature notes lower EGFR expression in HPV[+] vs. HPV[-] OPSCC. Yet, HPV oncoproteins E5, E6 and E7 each work to increase EGFR signaling, and MAPK-regulated AP-1 transcription factors direct HPV early gene expression. The goal of this work is to test the hypothesis that upon infection, HPV establishes a feed-forward loop with the EGFR pathway to drive viral gene expression independent of EGFR levels. We modeled early neoplasia with cell lines maintaining episomal HPV16 genomes, and cancers using SCC lines with integrated HPV16 genomes. Cells treated with EGF, EGFR inhibitors (cetuximab, erlotinib), or MEK antagonists (PD98059, trametinib) were evaluated for signaling and viral responses. Consistent with our hypothesis, we found increased EGF-dependent EGFR activation levels in HPV[+] cell lines, as well as heightened MEK1/2 activity independent of EGFR activation in HPV[+] cell lines compared to uninfected cells. RT-qPCR revealed that HPV oncogene transcription was enhanced by EGFR activation, whereas EGFR and MEK inhibitors led to significant declines in HPV transcription. In cells with episomal HPV genomes, lower viral genome levels accompanied inhibitor-reduced viral transcription. These data suggest EGFR/MAPK pathway interference leads to anti-viral affects, which are expected to restore p53 and pRb function and prevent tumorigenesis in vivo. As predicted, cell proliferation and tumor growth in NOD/SCID-gamma mouse HPV[+] SCC xenografts were significantly reduced with EGFR and MEK inhibitors. Experiments are underway to quantify the restored function of p53 and pRb in drug-treated cells and determine if these cells become sensitized to lower doses of cisplatin or radiation. In summary, our results reveal HPV infection creates a feed-forward loop with the EGFR/MAPK proliferation pathway, and EGFR and MEK antagonists have anti-viral effects, including reduction in the E6 and E7 oncoproteins needed to maintain the transformed phenotype in vitro and in vivo. Additionally, the ability of the drugs to reduce episomal viral genome levels suggests a potential curative effect if administered prior to viral genome integration. Lastly, this work reveals an anti-viral molecular mechanism which may account for the favorable outcomes of patients with HPV[+] OPSCC treated with cetuximab-radiotherapy. Citation Format: Anastacia M. Griego, Pamela Barraza, Chelin Hu, Agnieszka Dziduszko, Brianna K. Crowley, Helen J. Hathaway, Julie E. Bauman, Michelle A. Ozbun. The EGFR pathway as the Achilles’ heel for human papillomavirus-induced tumors: EGFR/MAPK pathway inhibitors exhibit antiviral activities and limit tumor growth in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3176. doi:10.1158/1538-7445.AM2014-3176
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