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Oral cancer causes significant global mortality and has a five‐year survival rate of around 64%. Poor prognosis results from late‐stage diagnosis, highlighting an important need to develop better approaches to detect oral premalignant lesions (OPLs) and identify which OPLs are at highest risk of progression to oral squamous cell carcinoma (OSCC). An appropriate animal model that reflects the genetic, histologic, immunologic, molecular and gross visual features of human OSCC would aid in the development and evaluation of early detection and risk assessment strategies. Here, we present an experimental PIK3CA + 4NQO transgenic mouse model of oral carcinogenesis that combines the PIK3CA oncogene mutation with oral exposure to the chemical carcinogen 4NQO, an alternate experimental transgenic mouse model with PIK3CA as well as E6 and E7 mutations, and an existing wild‐type mouse model based on oral exposure to 4NQO alone. We compare changes in dorsal and ventral tongue gross visual appearance, histologic features and molecular biomarker expression over a time course of carcinogenesis. Both transgenic models exhibit cytological and architectural features of dysplasia that mimic human disease and exhibit slightly increased staining for Ki‐67, a cell proliferation marker. The PIK3CA + 4NQO model additionally exhibits consistent lymphocytic infiltration, presents with prominent dorsal and ventral tongue tumours, and develops cancer quickly relative to the other models. Thus, the PIK3CA + 4NQO model recapitulates the multistep genetic model of human oral carcinogenesis and host immune response in carcinogen‐induced tongue cancer, making it a useful resource for future OSCC studies. more...

PURPOSE Pembrolizumab is a humanized monoclonal antibody that blocks interaction between programmed death receptor-1 (PD-1) and its ligands (PD-L1, PD-L2). Although pembrolizumab is approved for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC), its role in the management of locally advanced (LA) disease is not defined. We report a phase IB study evaluating the safety and efficacy of adding pembrolizumab to cisplatin-based chemoradiotherapy in patients with LA HNSCC. PATIENTS AND METHODS Eligible patients included those with oral cavity (excluding lip), oropharyngeal, hypopharyngeal, or laryngeal stage III to IVB HNSCC (according to American Joint Committee on Cancer, 7th edition, staging system) eligible for cisplatin-based, standard-dose (70 Gy) chemoradiotherapy. Pembrolizumab was administered concurrently with and after chemoradiotherapy with weekly cisplatin. Safety was the primary end point and was determined by incidence of chemoradiotherapy adverse events (AEs) and immune-related AEs (irAEs). Efficacy was defined as complete response (CR) rate on end-of-treatment (EOT) imaging or with pathologic confirmation at 100 days postradiotherapy completion. Key secondary end points included overall (OS) and progression-free survival (PFS). RESULTS The study accrued 59 patients (human papillomavirus [HPV] positive, n = 34; HPV negative, n = 25) from November 2015 to October 2018. Five patients (8.8%) required discontinuation of pembrolizumab because of irAEs, all of which occurred during concurrent chemoradiotherapy; 98.3% of patients completed the full planned treatment dose (70 Gy) of radiotherapy without any delays ≥ 5 days; 88.1% of patients completed the goal cisplatin dose of ≥ 200 mg/m2. EOT CR rates were 85.3% and 78.3% for those with HPV-positive and -negative HNSCC, respectively. CONCLUSION Pembrolizumab in combination with weekly cisplatin-based chemoradiotherapy is safe and does not impair delivery of curative radiotherapy or chemotherapy in HNSCC. Early efficacy data support further investigation of this approach. more...

Dysregulation of the Hippo signaling pathway and the consequent YAP1 activation is a frequent event in human malignancies, yet the underlying molecular mechanisms are still poorly understood. A pancancer analysis of core Hippo kinases and their candidate regulating molecules revealed few alterations in the canonical Hippo pathway, but very frequent genetic alterations in the FAT family of atypical cadherins. By focusing on head and neck squamous cell carcinoma (HNSCC), which displays frequent FAT1 alterations (29.8%), we provide evidence that FAT1 functional loss results in YAP1 activation. Mechanistically, we found that FAT1 assembles a multimeric Hippo signaling complex (signalome), resulting in activation of core Hippo kinases by TAOKs and consequent YAP1 inactivation. We also show that unrestrained YAP1 acts as an oncogenic driver in HNSCC, and that targeting YAP1 may represent an attractive precision therapeutic option for cancers harboring genomic alterations in the FAT1 tumor suppressor genes., Dysregulation of the Hippo signaling is a frequent event in human malignancies, but the molecular mechanisms remain unclear. Here the authors show that in head and neck squamous carcinoma, FAT1 interacts with the Hippo signaling complex, resulting in the activation of core Hippo kinases and YAP1 inactivation. more...

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Tobacco use is the main risk factor for HNSCC, and tobacco-associated HNSCCs have poor prognosis and response to available treatments. Recently approved anti-PD-1 immune checkpoint inhibitors showed limited activity (≤20%) in HNSCC, highlighting the need to identify new therapeutic options. For this, mouse models that accurately mimic the complexity of the HNSCC mutational landscape and tumor immune environment are urgently needed. Here, we report a mouse HNSCC model system that recapitulates the human tobacco-related HNSCC mutanome, in which tumors grow when implanted in the tongue of immunocompetent mice. These HNSCC lesions have similar immune infiltration and response rates to anti-PD-1 (≤20%) immunotherapy as human HNSCCs. Remarkably, we find that >70% of HNSCC lesions respond to intratumoral anti-CTLA-4. This syngeneic HNSCC mouse model provides a platform to accelerate the development of immunotherapeutic options for HNSCC., Tobacco use is one of the major risk factors for Head and neck squamous cell carcinoma (HNSCC). Here, the authors report an immune-competent syngeneic mouse model that mimics human tobacco-related HNSCC, and develops tumors in the tongue, and report a high response rate to anti-CTLA-4 therapy. more...

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Tobacco use is the main risk factor for HNSCC, and tobacco-associated HNSCCs have poor prognosis and response to available treatments. Recently approved anti-PD-1 immune checkpoint inhibitors showed limited activity (≤20%) in HNSCC, highlighting the need to identify new therapeutic options. For this, mouse models that accurately reflect the complexity of the HNSCC mutational landscape and tumor immune environment are urgently needed. Here, we report the first mouse HNSCC model system that recapitulates the human tobacco-related HNSCC mutanome, in which tumors grow when implanted in the tongue of immunocompetent mice. These HNSCC lesions have similar immune infiltration and response rates to anti-PD-1 (≤20%) immunotherapy as human HNSCCs. Remarkably, we found that >70% of HNSCC lesions respond to intratumoral anti-CTLA-4. This syngeneic HNSCC mouse model provides a platform for the development of novel immunotherapeutic options for HNSCC. more...

The rising incidence of human papillomavirus (HPV)-associated malignancies, especially for oropharyngeal cancers, has highlighted the urgent need to understand how the interplay between high-risk HPV oncogenes and carcinogenic exposure results in squamous cell carcinoma (SCC) development. Here, we describe an inducible mouse model expressing high risk HPV-16 E6/E7 oncoproteins in adults, bypassing the impact of these viral genes during development. HPV-16 E6/E7 genes were targeted to the basal squamous epithelia in transgenic mice using a doxycycline inducible cytokeratin 5 promoter (cK5-rtTA) system. After doxycycline induction, both E6 and E7 were highly expressed, resulting in rapid epidermal hyperplasia with a remarkable expansion of the proliferative cell compartment to the suprabasal layers. Surprisingly, in spite of the massive growth of epithelial cells and their stem cell progenitors, HPV-E6/E7 expression was not sufficient to trigger mTOR activation, a key oncogenic driver in HPV-associated malignancies, and malignant progression to SCC. However, these mice develop SCC rapidly after a single exposure to a skin carcinogen, DMBA, which was increased by the prolonged exposure to a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Thus, only few oncogenic hits may be sufficient to induce cancer in E6/E7 expressing cells. All HPV-E6/E7 expressing SCC lesions exhibited increased mTOR activation. Remarkably, rapamycin, an mTOR inhibitor, abolished tumor development when administered to HPV-E6/E7 mice prior to DMBA exposure. Our findings revealed that mTOR inhibition protects HPV-E6/E7 expressing tissues form SCC development upon carcinogen exposure, thus supporting the potential clinical use of mTOR inhibitors as a molecular targeted approach for prevention of HPV-associated malignancies. more...

Abstract The incidence of human papillomavirus-associated head and neck squamous cell carcinoma (HPV[ + ] HNSCC) is rapidly increasing. Although clinical management of primary HPV( + ) HNSCC is relatively successful, disease progression, including recurrence and metastasis, is often fatal. Moreover, patients with progressive disease face limited treatment options and significant treatment-associated morbidity. These clinical data highlight the need to identify targetable mechanisms that drive disease progression in HPV( + ) HNSCC to prevent and/or treat progressive disease. Interestingly, β-adrenergic signaling has recently been associated with pro-tumor processes in several disease types. Here we show that an aggressive murine model of recurrent/metastatic HPV( + ) HNSCC upregulates β2-adrenergic receptor (β2AR) expression, concordant with significantly heightened mitochondrial metabolism, as compared with the parental model from which it spontaneously derived. β-Adrenergic blockade effectively inhibits in vitro proliferation and migratory capacity in this model, effects associated with an attenuation of hyperactive mitochondrial respiration. Importantly, propranolol, a clinically available nonselective β-blocker, significantly slows primary tumor growth, inhibits metastatic development, and shows additive benefit alongside standard-of-care modalities in vivo. Further, via CRISPR/Cas9 technology, we show that the hyperactive mitochondrial metabolic profile and aggressive in vivo phenotype of this recurrent/metastatic model are dependent on β2AR expression. These data implicate β2AR as a modulator of mitochondrial metabolism and disease progression in HPV( + ) HNSCC, and warrant further investigation into the use of β-blockers as low cost, relatively tolerable, complementary treatment options in the clinical management of this disease. more...

Oral mucosal wound healing has long been regarded as an ideal system of wound resolution. However, the intrinsic characteristics that mediate optimal healing at mucosal surfaces are poorly understood, particularly in humans. We present a unique comparative analysis between human oral and cutaneous wound healing using paired and sequential biopsies during the repair process. Using molecular profiling, we determined that wound-activated transcriptional networks are present at basal state in the oral mucosa, priming the epithelium for wound repair. We show that oral mucosal wound-related networks control epithelial cell differentiation and regulate inflammatory responses, highlighting fundamental global mechanisms of repair and inflammatory responses in humans. The paired comparative analysis allowed for the identification of differentially expressed SOX2 (sex-determining region Y-box 2) and PITX1 (paired-like homeodomain 1) transcriptional regulators in oral versus skin keratinocytes, conferring a unique identity to oral keratinocytes. We show that SOX2 and PITX1 transcriptional function has the potential to reprogram skin keratinocytes to increase cell migration and improve wound resolution in vivo. Our data provide insights into therapeutic targeting of chronic and nonhealing wounds based on greater understanding of the biology of healing in human mucosal and cutaneous environments. more...

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. There is an urgent need to develop effective therapeutic approaches to prevent and treat HNSCC. Recent deep sequencing of the HNSCC genomic landscape revealed a multiplicity and diversity of genetic alterations in this malignancy. Although a large variety of specific molecules were found altered in each individual tumor, they all participate in only a handful of driver signaling pathways. Among them, the PI3K/mTOR pathway is the most frequently activated, which plays a central role in cancer initiation and progression. In turn, targeting of mTOR may represent a precision therapeutic approach for HNSCC. Indeed, mTOR inhibition exerts potent anti-tumor activity in HNSCC experimental systems, and mTOR targeting clinical trials show encouraging results. However, advanced HNSCC patients may exhibit unpredictable drug resistance, and the analysis of its molecular basis suggests that co-targeting strategies may provide a more effective option. In addition, although counterintuitive, emerging evidence suggests that mTOR inhibition may enhance the anti-tumor immune response. These new findings raise the possibility that the combination of mTOR inhibitors and immune oncology agents may provide novel precision therapeutic options for HNSCC. more...

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Tobacco use is the main risk factor for HNSCC, and tobacco-associated HNSCCs have poor prognosis and response to available treatments. Recently approved anti-PD-1 immune checkpoint inhibitors showed limited activity (≤20%) in HNSCC, highlighting the need to identify new therapeutic options. For this, mouse models that accurately reflect the complexity of the HNSCC mutational landscape and tumor immune environment are urgently needed. Here, we report the first mouse HNSCC model system that recapitulates the human tobacco-related HNSCC mutanome, in which tumors grow when implanted in the tongue of immunocompetent mice. These HNSCC lesions have similar immune infiltration and response rates to anti-PD-1 (≤20%) immunotherapy as human HNSCCs. Remarkably, we found that >70% of HNSCC lesions respond to intratumoral anti-CTLA-4. This syngeneic HNSCC mouse model provides a platform for the development of novel immunotherapeutic options for HNSCC. Note:This abstract was not presented at the conference. Citation Format: Zhiyong Wang, Victoria H. Wu, Michael M. Allevato, Mara Gilardi, Robert Saddawi-Konefka, Yudou He, Juan Luis Callejas-Valera, Lynn Vitale-Cross, Daniel Martin, Panomwat Amornphimoltham, James Mcdermott, Yusuke Goto, Alfredo A. Molinolo, Andrew B. Sharabi, Ezra E.W. Cohen, Qianming Chen, Guy Lyons, Ludmil B. Alexandrov, J. Silvio Gutkind. Novel syngeneic animal model of tobacco-associated oral cancer reveals the activity of in situ anti-CTLA-4 [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B51. more...

G-protein coupled receptors (GPCRs), which activate heterotrimeric G proteins, are an essential class of transmembrane receptors that are responsible for a myriad of signaling events in normal and pathologic conditions. Two members of the G protein family, Gαq and Gα11, activate one of the main GPCR pathways and function as oncogenes by integrating mitogen-stimulated signaling cascades that are active under malignant conditions. Recently, it has been shown that targeted deletion of Gα11 and Gαq from endothelial cells impairs the Rho-mediated formation of focal adherens junctions, suggesting that Gα11/q signaling may also play a significant role in cytoskeletal-mediated cellular responses in epithelial cells. Indeed, combined deletion of Gα11 and Gαq confers a significant migratory defect in keratinocytes that delays cutaneous wound healing in an in vivo setting. This delay can be attributed to a defect during the reepithelialization phase due to significantly attenuated migratory capacity of Gαq-null keratinocytes under combined Gα11 deficiency. In fact, cells lacking Gα11/q demonstrate a severely reduced ability to respond to mitogenic and migratory signals in the microenvironment, leading to inappropriate and premature terminal differentiation. These results suggest that Gα11/q signaling pathways may be critical for integrating mitogenic signals and cytoskeletal function to achieve normal physiological responses. Emergence of a malignant phenotype may therefore arise from both under- and overexpression of Gα11/q signaling, implicating its upstream regulation as a potential therapeutic target in a host of pathologic conditions. more...

The E1a gene from adenovirus has become a major tool in cancer research. Since the discovery of E1a, it has been proposed to be an oncogene, becoming a key element in the model of cooperation between oncogenes. However, E1a's in vivo behaviour is consistent with a tumour suppressor gene, due to the block/delay observed in different xenograft models. To clarify this interesting controversy, we have evaluated the effect of the E1a 13s isoform from adenovirus 5 in vivo. Initially, a conventional xenograft approach was performed using previously unreported HCT116 and B16-F10 cells, showing a clear anti-tumour effect regardless of the mouse's immunological background (immunosuppressed/immunocompetent). Next, we engineered a transgenic mouse model in which inducible E1a 13s expression was under the control of cytokeratin 5 to avoid side effects during embryonic development. Our results show that E1a is able to block chemical skin carcinogenesis, showing an anti-tumour effect. The present report demonstrates the in vivo anti-tumour effect of E1a, showing that the in vitro oncogenic role of E1a cannot be extrapolated in vivo, supporting its future use in gene therapy approaches., This work was supported by grants from Fundación Leticia Castillejo Castillo and MINECO (SAF 2015 64215-R) and from JCCM (PPII10-0141-0404) to RSP. F. Cimas was supported by Fundación Leticia Castillejo Castillo and Cátedra Enresa. RSP research institution received support from the European Community through the regional development funding programme (FEDER). more...

5-Fluorouracil (5-FU), together with other drugs such as oxaliplatin, is one of the most important pharmacological agents in the treatment of colorectal cancer. Although mitogen-activated protein kinases (MAPKs) have been extensively connected with resistance to platinum compounds, no role has been established in 5-FU resistance. Here we demonstrate that p38MAPK activation is a key determinant in the cellular response to 5-FU. Thus, inhibition of p38MAPKα by SB203580 compound or by short-hairpin RNA interference-specific knockdown correlates with a decrease in the 5-FU-associated apoptosis and chemical resistance in both HaCaT and HCT116 cells. Activation of p38MAPK by 5-FU was dependent on canonical MAP2K, MAPK kinase (MKK)-3 and MKK6. In addition, ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) showed a redundancy of function for the final activation of p38MAPK. Resistance associated with p38MAPK inhibition correlates with an autophagic response that was mediated by a decrease in p53-driven apoptosis, without effect onto p53-dependent autophagy. Moreover, the results with colorectal cancer-derived cell lines with different p53 status and patterns of resistance to 5-FU suggest that de novo and acquired resistance was controlled by similar mechanisms. In summary, our data demonstrate a critical role for the p38MAPK signaling pathway in the cellular response to 5-FU by controlling the balance between apoptosis and autophagy. more...

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In response to oncogenic signals, cells have developed safe mechanisms to avoid transformation through activation of a senescence program. Upon v-H-Ras overexpression, normal cells undergo senescence through several cellular processes, including activation of the ERK1/2 pathway. Interestingly, the E1a gene from adenovirus 5 has been shown to rescue cells from senescence by a yet unknown mechanism. We investigated whether E1a was able to interfere with the ERK1/2 signaling pathway to rescue cells from v-H-Ras-mediated senescence. Our results show that, E1a overexpression blocks v-H-Ras-mediated ERK1/2 activation by two different and concomitant mechanisms. E1a through its ability to interfere with PKB/Akt activation induces the down-regulation of the PEA15 protein, an ERK1/2 nuclear export factor, leading to nuclear accumulation of ERK1/2. In addition to this, we show that E1a increases the expression of the inducible ERK1/2 nuclear phosphatases (MAPK phosphatases) MKP1/DUSP1 and DUSP5, which leads to ERK1/2 dephosphorylation. We confirmed our observations in the human normal diploid fibroblasts IMR90, in which we could also show that an E1a mutant, unable to bind retinoblastoma protein (pRb), cannot rescue cells from v-H-Ras-induced senescence. In conclusion, E1a is able to rescue from Ras-induced senescence by affecting ERK1/2 localization and phosphorylation. more...

Activation of p38 MAPK is a critical requisite for the therapeutics activity of the antitumor agent cisplatin. In this sense, a growing body of evidences supports the role of c-Abl as a major determinant of p38 MAPK activation, especially in response to genotoxic stress when triggered by cisplatin. Here, we demonstrate that p38 MAPK activation in response to cisplatin does not require the tyrosine kinase activity of c-Abl. Indeed, c-Abl can activate the p38 MAPK signaling pathway by a mechanism that is independent of its tyrosine kinase activity, but that instead involves the ability of c-Abl to increase the stability of MKK6. Similar results were obtained in chronic myeloid leukemia-derived cell lines, in which a chimeric Bcr/Abl protein mimics the effects of c-Abl overexpression on p38 MAPK activation. These findings may explain why a clinically used c-Abl inhibitor, imatinib mesylate, fails to inhibit the p38 MAPK pathway alone or in combination with cisplatin, and provide evidence of a novel signaling mechanism in which these antitumor agents act. more...

// Francisco J. Cimas 1, * , Juan L. Callejas-Valera 2, * , Raquel Pascual-Serra 1 , Jesus Garcia-Cano 1 , Elena Garcia-Gil 1 , Miguel De la Cruz-Morcillo 1 , Marta Ortega-Muelas 1 , Leticia Serrano-Oviedo 1 , J. Silvio Gutkind 2 , Ricardo Sanchez-Prieto 1 1 Unidad de Medicina Molecular, laboratorio de Oncologia, Centro Regional de Investigaciones Biomedicas, Universidad de Castilla-La Mancha, 02006, Albacete, Spain Unidad de Biomedicina UCLM-CSIC 2 Moores Cancer Center/UCSD, La Jolla, CA 92093-0819, USA * These authors contributed equally to this work Correspondence to: Ricardo Sanchez-Prieto, e-mail: ricardo.sanchez@uclm.es Juan L. Callejas-Valera, e-mail: jlcallejasvalera@ucsd.edu Keywords: E1a, MKP1, cisplatin, chemotherapy, lung cancer Received: September 02, 2015 Accepted: November 25, 2015 Published: December 12, 2015 ABSTRACT The adenoviral gene E1a is known to enhance the antitumor effect of cisplatin, one of the cornerstones of the current cancer chemotherapy. Here we study the molecular basis of E1a mediated sensitivity to cisplatin in an experimental model of Non-small cell lung cancer. Our data show how E1a blocks the induction of autophagy triggered by cisplatin and promotes the apoptotic response in resistant cells. Interestingly, at the molecular level, we present evidences showing how the phosphatase MKP1 is a major determinant of cisplatin sensitivity and its upregulation is strictly required for the induction of chemosensitivity mediated by E1a. Indeed, E1a is almost unable to promote sensitivity in H460, in which the high expression of MKP1 remains unaffected by E1a. However, in resistant cell as H1299, H23 or H661, which display low levels of MKP1, E1a expression promotes a dramatic increase in the amount of MKP1 correlating with cisplatin sensitivity. Furthermore, effective knock down of MKP1 in H1299 E1a expressing cells restores resistance to a similar extent than parental cells. In summary, the present work reinforce the critical role of MKP1 in the cellular response to cisplatin highlighting the importance of this phosphatase in future gene therapy approach based on E1a gene. more...

The rising incidence of HPV-associated malignancies, especially for head and neck squamous cell carcinoma (HNSCC), has highlighted the urgent need to understand the role of HPV-16 E6/E7 oncogenes in cancer initiation and progression. For that propose we decided to generate a doxycycline inducible E6/E7 transgenic mouse targeting E6/E7 onco-proteins to the basal epithelia layer, including the stem cell compartment, through a doxycycline inducible cytokeratin 5 promoter (cK5-rtTA). This system allows to turn on the expression of HPV genes after birth, minimizing the negative effect that viral E6/E7 expression may have during embryonic development, and mimicking the natural infection process that usually happens in adults after initiation of sexual activities. Interestingly, we found that after doxycycline induction, both E6 and E7 were highly expressed, resulting in a clear increase in the number of proliferative cells even in the suprabasal layers, which results in rapid epidermal hyperplasia. However, in our animal mouse model HPV-16 E6/E7 expression alone was not sufficient to induce spontaneous squamous carcinomas (SCC). Due that we decided to challenge the oncogenic role of HPV using a classical chemical carcinogenesis protocol (DMBA-TPA). These mice develop SCC rapidly after a single exposure to a skin carcinogen, DMBA, which was increased by the prolonged exposure to a tumor promoter, TPA. These data suggest that only few oncogenic hits may be sufficient to induce cancer in humans already infected with HPV-16 E6/E7. Then, we decided to elucidate whether or not a few genomic alterations in combination with HPV-16 expression may be sufficient to induce cancer. One of the most frequent mutations in HNSCC, including HPV-associated cancers, are those affecting the PI3K-mTOR pathway. Based on that, we engineered a genetically defined mouse model combining HPV-16 E6/E7 and PIk3CAH1047R expression under the control of a cytokeratin 14 promoter driven tamoxifen-regulated Cre recombinase (cK14-CreERTM). Surprisingly, we found that upon tamoxifen activation mice develop oral cancer in only few weeks. Furthermore, our HPV-related cancer mouse models exhibited a marker increase of mTOR activation which is a key oncogenic driver in malignant progression to SCC. Remarkably, rapamycin, an mTOR inhibitor, was able to block tumor development, supporting the potential clinical use of direct and indirect mTOR inhibitors as a molecular targeted approach for prevention of HPV-associated malignancies. Citation Format: Juan Luis Callejas-Valera, Ramiro Iglesias-Bartolome, Panomwat Amornphimoltham, Julia Palacios-Garcia, Daniel Martin, Joseph A. Califano, Alfredo A. Molinolo, J.Silvio Gutkind. A next-gen animal model to Study PIK3CA-mTOR driven HPV-related oral malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4800. doi:10.1158/1538-7445.AM2017-4800 more...

MAP Kinase Phosphatase 1 (MKP1) - also known as Dual Specificity Phosphatase 1 (DUSP1) or CL100 - is a known member of Serine/Threonine inducible nuclear phosphatase family. Interestingly, this subset of phosphatases acts as MAPKs counterpart allowing switching off MAPKs as p38MAPK, ERK1/2, ERK5 or JNK. It is well-known that MKP1 plays critical roles in different key biological processes ranging from the immune system up to cell differentiation. On the other hand, it is also known that MKP1 levels are altered in several tumours, such as bladder, ovarian, prostate, breast, colon, and non-small cell lung cancers (NSCLC) among others (for a review of MKP1 and cancer see [1]). For example, in ovarian cancer, high MKP1 expression levels correlate with a clear reduction in tumour recurrence-free survival [2]. In addition, it is also reported that MKP1 levels are highly upregulated after cisplatin treatment in ovarian cancer, and MKP1 knockdown promotes cisplatin mediated apoptosis in the same pathology [3]. Indeed, a similar role has been proposed in lung or breast cancer. Therefore, MKP1 was considered to confer oncogenic and chemo-resistance properties, suggesting that down-modulation could be a good therapeutic approach. However, recent evidences indicate that MKP1 is a potential inducer of chemo-sensitivity in a very special context such as the presence of E1a gene. E1A is the first transcript of the early region of the adenovirus which is a key part of the infective cycle. It is responsible for regulating the transcription of the later adenoviral genes, promoting the entrance in S phase and the suppression of the defensive mechanisms of the host cell. E1A expression is also responsible for apoptosis induction in different experimental models, being able to collaborate in chemo and radiotherapy treatments. Recently, we observed that E1A was able to increase cisplatin sensitivity in some resistant NSCLC cell lines (as H1299) through MKP1 transcriptional upregulation mediated by E1A expression [4]. Indeed, MKP1 knockdown restores chemo resistance in E1A expressing H1299 cell lines, confirming how the axis E1A -> MKP1 -> p38MAPK promotes sensitivity, blocking the characteristic autophagic response associated to resistant models [5], and promoting an increase in the apoptotic onset. Indeed, inhibition of p38 has been also recently proposed as a mechanism of chemosensitivity in different in vitro and in vivo models [6]. Therefore, all these observations should be considered in a future therapy based in MKP1 inhibition. In this regard the obvious question is what will happen in a tumour in which the transformation is based in an E1A-like mechanism. It is clear that in this case MKP1 inhibition will promote resistance to conventional chemotherapy, as is the case of cisplatin. So, the next question is to know if an E1A-like mechanism is implicated in tumorigenesis. In this regard, we demonstrated several years ago how E1A was able to bypass Oncogene-Induced Senescence (OIS), as the induced by v-H-Ras, increasing MKP1 transcriptional levels, enabling in this way the nuclear inactivation of ERK1/2 [7]. Therefore, as the scape from OIS is a key event in cellular transformation, it seems to be highly probable that an E1A-like mechanism could be implicated. Therefore, while MKP1 is a villain (oncogenic) in E1A mediated transformation, it could be also the good Samaritan able to promote chemosensitivity in resistant tumours. However, the important question goes beyond the experimental model based in E1A: should we use the inhibition of MKP1 in cancer therapy? We do strongly believe that MKP1 is a novel biomarker and a possible therapeutic target, but this observation could not be applied indiscriminately to any tumour. The presence of specific alterations could be implicated in the loss of the therapeutic benefits associated to MKP1 inhibition, as it may be the presence of a physiologic context similar to the one observed in the presence of E1A. In fact, it is noteworthy that alteration in key proteins present in many tumours, like p400, p300 or pRb, are also targeted by E1A to exert its transforming properties. In sum, MKP1 have two (or even more) faces, but we should manage to use the one closer to Dr. Jekyll and get rid of Mr. Hyde to fully explore its therapeutic potential. more...

et al., The E1a gene from adenovirus is known to be a potent inducer of chemo/radiosensitivity in a wide range of tumors. However, the molecular bases of its radiosensitizer properties are still poorly understood. In an attempt to study this effect, U87MG cells, derived from a radio-resistant tumor as glioblastoma, where infected with lentivirus carrying E1a gene developing an acute sensitivity to ionizing radiation. The induction of radiosensitivity correlated with a marked G2/M phase accumulation and a potent apoptotic response. Our findings demonstrate that c-Myc plays a pivotal role in E1a-associated radiosensitivity through the induction of a replicative stress situation, as our data support by genetic approaches, based in interference and overexpression in U87MG cells. In fact, we present evidence showing that Chk1 is a novel transcriptional target of E1a gene through the effect exerted by this adenoviral protein onto c-Myc. Moreover, c-Myc upregulation also explains the marked phosphorylation of H2AX associated to E1a expression in the absence of DNA damage. Indeed, all these observations were applicable to other experimental models, such as T98G, LN-405 and A172, rendering the same pattern in terms of radiosensitivity, cell cycle distribution, upregulation of Chk1, c-Myc, and phosphorylation pattern of H2AX. In summary, our data propose a novel mechanism to explain how E1a mediates radiosensitivity through the signaling axis E1a→c-Myc→ replicative stress situation. This novel mechanism of E1a-mediated radiosensitivity could be the key to open new possibilities in the current therapy of glioblastoma., This work was supported by grants from Fundación Leticia Castillejo Castillo, JCCM (PPII10-0141-040) and Ministerio de Economia y Competitividad (SAF2012-30862) to RSP. Instituto de Salud Carlos III FIS PS09/1988 and CCG10-UAM/BIO-5871 to ISP. RSP Research Institute, and the work performed in his laboratory receive support from the European Community through the regional development funding program (FEDER). more...

The squamous epithelium covering the skin and oral mucosa relies on epithelial stem cells for tissue renewal. Dynamic changes in DNA methylation, histone methylation and acetylation, and higher order chromatin structure are required to preserve their self-renewal capacity while orchestrating the timely execution of cell differentiation programs. This complex network of epigenetic modifications shapes the epithelial stem cell identity and fate. Pathological alterations can be perceived by aberrant chromatin sensors, such as the INK4/ARF locus, which initiate tumor suppressive cell senescence programs, and can often result in epithelial stem cell exhaustion. Unveiling the mechanisms controlling the epigenome in epithelial stem cells may help protect against the loss of their tissue regenerative capacity, thereby preventing premature aging without increasing cancer risk. more...

The p38 MAPK signaling pathway has been proposed as a critical mediator of the therapeutic effect of several antitumor agents, including cisplatin. Here, we found that sensitivity to cisplatin, in a system of 7 non-small cell lung carcinoma derived cell lines, correlated with high levels of MKK6 and marked activation of p38 MAPK. However, knockdown of MKK6 modified neither the response to cisplatin nor the activation of p38 MAPK. Deeper studies showed that resistant cell lines also displayed higher basal levels of MKK3. Interestingly, MKK3 knockdown significantly decreased p38 phosphorylation upon cisplatin exposure and consequently reduced the response to the drug. Indeed, cisplatin poorly activated MKK3 in resistant cells, while in sensitive cell lines MKK3 showed the opposite pattern in response to the drug. Our data also demonstrate that the low levels of MKK6 expressed in resistant cell lines are the consequence of high basal activity of p38 MAPK mediated by the elevated levels of MKK3. This finding supports the existence of a regulatory mechanism between both MAPK kinases through their MAPK. Furthermore, our results were also mirrored in head and neck carcinoma derived cell lines, suggesting our observations boast a potential universal characteristic in cancer resistance of cisplatin. Altogether, our work provides evidence that MKK3 is the major determinant of p38 MAPK activation in response to cisplatin and, hence, the resistance associated with this MAPK. Therefore, these data suggest that the balance between both MKK3 and MKK6 could be a novel mechanism which explains the cellular response to cisplatin. more...

10 páginas, 6 figuras.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Resistance to Imatinib Mesylate (IM) is a major problem in Chronic Myelogenous Leukaemia management. Most of the studies about resistance have focused on point mutations on BCR/ABL. However, other types of resistance that do not imply mutations in BCR/ABL have been also described. In the present report we aim to study the role of several MAPK in IM resistance not associate to BCR/ABL mutations. Therefore we used an experimental system of resistant cell lines generated by co-culturing with IM (K562, Lama 84) as well as primary material from resistant and responder patient without BCR/ABL mutations. Here we demonstrate that Erk5 and p38MAPK signaling pathways are not implicated in the acquired resistance phenotype. However, Erk2, but not Erk1, is critical for the acquired resistance to IM. In fact, Bcr/Abl activates preferentially Erk2 in transient transfection in a dose dependent fashion through the c-Abl part of the chimeric protein. Finally, we present evidences demonstrating how constitutive activation of Erk2 is a de novo mechanism of resistance to IM. In summary our data support the use of therapeutic approaches based on Erk2 inhibition, which could be added to the therapeutic armamentarium to fight CML, especially when IM resistance develops secondary to Erk2 activation., This work was supported by grants from Fundación Mutua Madrileña del Automóvil, Fundación Leticia Castillejo Castillo and Ministerio de Educación y Ciencia. (SAF/2006/0179 and BFU2006-01813/BMC). RSP and AP Research Institutes, and the work carried out in their laboratories receive support from the European Community through the regional development funding program (FEDER). AEO is supported by the ISCIII (PI061552 and CP04/00045 contract). more...

G-protein coupled receptors (GPCRs) and G-proteins are critical signal transduction molecules that regulate cell survival, proliferation, motility and differentiation. However, mutations and disruptions in the expression or function of these molecules have been linked to growth, progression and metastasis of various cancers. Recently, major efforts in the area of genome-wide sequencing of tumors have unveiled numerous previously uncharacterized mutations in G-proteins and GPCRs, which could have significant implications to cancer initiation and progression. In particular, mutations in the gene encoding the G-protein G13 (GNA13) and/or its downstream effector, RhoA, have been identified in B cell lymphomas (particularly Burkitt's lymphoma and Diffuse Large B cell Lymphoma), T cell lymphomas, and Head and Neck Squamous Cell Carcinomas (HNSCCs). Our analyses indicate that these mutations are highly statistically significant over background cancer gene mutation rate, and mutations in GNA13 alone occur in ∼10-15% of patients with Burkitt's lymphoma and Diffuse Large B cell Lymphoma. Surprisingly, although G13 and RhoA have previously been linked to cellular transformation and metastatic potential of epithelial cancers, our data suggests that the mutations in GNA13 and/or RHOA in these B cell lymphomas and HNSCC are inhibitory in nature. Furthermore, our results indicate a tumor suppressive function for wild-type GNA13 and RHOA in in vivo cancer models. Overall, our data suggest a novel function tumor suppressive function for the G13/RhoA axis in multiple human malignancies. Citation Format: Morgan O'hayre, Asuka Inoue, Katiuchia Sales, Irina Kufareva, Juan Luis Callejas Valera, Fukun Guo, Constantinos Mikelis, Giovanni DiPasquale, Kira Finkel, Junken Aoki, Yi Zheng, Thomas H. Bugge, J. Silvio Gutkind. Novel roles for GNA13 and RHOA as tumor suppressor genes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2059. doi:10.1158/1538-7445.AM2015-2059 more...

mTOR inhibition has emerged as a promising strategy for head and neck squamous cell carcinomas (HNSCC) treatment. However, most targeted therapies ultimately develop resistance due to the activation of adaptive survival signaling mechanisms limiting the activity of targeted agents. Thus, co-targeting key adaptive mechanisms may enable more effective cancer cell killing. Here, we performed a synthetic lethality screen using shRNA libraries to identify druggable candidates for combinatorial signal inhibition. We found that the ERK pathway was the most highly represented. Combination of rapamycin with trametinib, a MEK1/2 inhibitor, demonstrated strong synergism in HNSCC-derived cells in vitro and in vivo, including HNSCC cells expressing the HRAS and PIK3CA oncogenes. Interestingly, cleaved caspase-3 was potently induced by the combination therapy in PIK3CA+ cells in vitro and tumor xenografts. Moreover, ectopic expression of PIK3CA mutations into PIK3CA- HNSCC cells sensitized them to the pro-apoptotic activity of the combination therapy. These findings indicate that co-targeting the mTOR/ERK pathways may provide a suitable precision strategy for HNSCC treatment. Moreover, PIK3CA+ HNSCC are particularly prone to undergo apoptosis after mTOR and ERK inhibition, thereby providing a potential biomarker of predictive value for the selection of patients that may benefit from this combination therapy. more...