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1.  Heparin inhibits Angiotensin II-induced vasoconstriction on isolated mouse mesenteric resistance arteries through Rho-A- and PKA-dependent pathways 
Vascular pharmacology  2012;58(4):313-318.
Heparin is commonly used to treat intravascular thrombosis in children undergoing extracorporeal membrane oxygenation or cardiopulmonary bypass. These clinical circumstances are associated with elevated plasma levels of angiotensin II (Ang II). However, the mechanisms by which heparin modulates vascular reactivity of Ang II remain unclear. We hypothesized that heparin may offset Ang II-induced vasoconstriction on mesenteric resistances arteries through modulating the Rho-A/Rho kinase pathway. Vascular contractility was studied using pressurized, resistance-sized mesenteric arteries from mice. Rho-A activation was measured by pull-down assay, and myosin light chain or PKA phosphorylation by immunoblotting. We found that heparin significantly attenuated vasoconstriction induced by Ang II but not that by KCl. The combined effect of Ang II with heparin was almost abolished by a specific Rho kinase inhibitor Y27632. Ang II stimulated Rho-A activation and myosin light chain phosphorylation, both responses were antagonized by heparin. Moreover, the inhibitory effect of heparin on Ang II-induced vasoconstriction was reversed by Rp-cAMPS (cAMP-dependent PKA inhibitor), blunted by ODQ (soluble guanylate cyclase inhibitor), and mimicked by a cell-permeable cGMP analogue, 8-Br-cGMP, but not by a cAMP analogue. PKC and Src kinase were not involved. We conclude that heparin inhibits Ang II-induced vasoconstriction through Rho-A/Rho kinase- and cGMP/PKA-dependent pathways.
doi:10.1016/j.vph.2012.12.003
PMCID: PMC3606668  PMID: 23268358
Ang II; heparin; resistance arteries; Rho-A/Rho kinase; vascular tone
2.  ARF1 regulates the Rho/MLC pathway to control EGF-dependent breast cancer cell invasion 
Molecular Biology of the Cell  2014;25(1):17-29.
The small GTPase ARF1 is overexpressed in invasive breast cancer cells. This ARF isoform controls MMP-9 activity to degrade the extracellular matrix by regulating invadopodia maturation and microvesicle shedding. The molecular mechanisms by which ARF1 controls invasiveness involve regulation of the Rho/MLC pathway.
Invasion of tumor cells is a key step in metastasis that depends largely on the ability of these cells to degrade the extracellular matrix. Although we have showed that the GTPase ADP-ribosylation factor 1 (ARF1) is overexpressed in highly invasive breast cancer cell lines and that epidermal growth factor stimulation can activate this ARF isoform to regulate migration as well as proliferation, the role of this small GTP-binding protein has not been addressed in the context of invasiveness. Here we report that modulation of ARF1 expression and activity markedly impaired the ability of M.D. Anderson-metastatic breast-231 cells, a prototypical highly invasive breast cancer cell line, to degrade the extracellular matrix by controlling metalloproteinase-9 activity. In addition, we demonstrate that this occurs through inhibition of invadopodia maturation and shedding of membrane-derived microvesicles, the two key structures involved in invasion. To further define the molecular mechanisms by which ARF1 controls invasiveness, we show that ARF1 acts to modulate RhoA and RhoC activity, which in turn affects myosin light-chain (MLC) phosphorylation. Together our findings underscore for the first time a key role for ARF1 in invasion of breast cancer cells and suggest that targeting the ARF/Rho/MLC signaling axis might be a promising strategy to inhibit invasiveness and metastasis.
doi:10.1091/mbc.E13-06-0335
PMCID: PMC3873888  PMID: 24196838
3.  Rab25 regulates invasion and metastasis in head and neck cancer 
Purpose
Head and neck squamous cell carcinoma (HNSCC) is one of the ten most common cancers with a 50% five-year survival rate, which has remained unchanged for the past three decades. One of the major reasons for the aggressiveness of this cancer is that HNSCCs readily metastasize to cervical lymph nodes that are abundant in the head and neck region. Hence, discovering new molecules controlling the metastatic process as well as understanding their regulation at the molecular level are essential for effective therapeutic strategies.
Experimental Design
Rab25 expression level was analyzed in HNSCC tissue microarray. We used a combination of intravital microscopy in live animals and immunofluorescence in an in vitro invasion assay, to study role of Rab25 in tumor cells migration and invasion.
Results
In this study, we identified the small GTPase Rab25 as a key regulator of HNSCC metastasis. We observed that Rab25 is downregulated in HNSCC patients. Next, we determined that re-expression of Rab25 in a metastatic cell line is sufficient to block invasion in a 3D collagen matrix and metastasis to cervical lymph nodes in a mouse model for oral cancer. Specifically, Rab25 affects the organization of F-actin at the cell surface, rather than cell proliferation, apoptosis or tumor angiogenesis.
Conclusion
These findings suggest that Rab25 plays an important role in tumor migration and metastasis, and that understanding its function may lead to the development of new strategies to prevent metastasis in oral cancer patients.
doi:10.1158/1078-0432.CCR-12-2858
PMCID: PMC3602237  PMID: 23340300
Rab25; metastasis; oral cancer; intravital microscopy; actin cytoskeleton
5.  DSG3 as a Biomarker for the Ultrasensitive Detection of Occult Lymph Node Metastasis in Oral Cancer Using Nanostructured Immunoarrays 
Oral oncology  2012;49(2):93-101.
Objectives
The diagnosis of cervical lymph node metastasis in head and neck squamous cell carcinoma (HNSCC) patients constitutes an essential requirement for clinical staging and treatment selection. However, clinical assessment by physical examination and different imaging modalities, as well as by histological examination of routine lymph node cryosections can miss micrometastases, while false positives may lead to unnecessary elective lymph node neck resections. Here, we explored the feasibility of developing a sensitive assay system for desmoglein 3 (DSG3) as a predictive biomarker for lymph node metastasis in HNSCC.
Materials and Methods
DSG3 expression was determined in multiple general cancer- and HNSCC-tissue microarrays (TMA), in negative and positive HNSCC metastatic cervical lymph nodes, and in a variety of HNSCC and control cell lines. A nanostructured immunoarray system was developed for the ultrasensitive detection of DSG3 in lymph node tissue lysates.
Results
We demonstrate that DSG3 is highly expressed in all HNSCC lesions and their metastatic cervical lymph nodes, but absent in non-invaded lymph nodes. We show that DSG3 can be rapidly detected with high sensitivity using a simple microfluidic immunoarray platform, even in human tissue sections including very few HNSCC invading cells, hence distinguishing between positive and negative lymph nodes.
Conclusion
We provide a proof of principle supporting that ultrasensitive nanostructured assay systems for DSG3 can be exploited to detect micrometastatic HNSCC lesions in lymph nodes, which can improve the diagnosis and guide in the selection of appropriate therapeutic intervention modalities for HNSCC patients.
doi:10.1016/j.oraloncology.2012.08.001
PMCID: PMC3546158  PMID: 23010602
DSG3; Head and Neck Cancer; Desmosomes; Biomarker; Sentinel Lymph Nodes; Nanosensors
6.  Rapid Microfluidic Immunoassays of Cancer Biomarker Proteins Using Disposable Inkjet-Printed Gold Nanoparticle Arrays 
ChemistryOpen  2013;2(4):141-145.
doi:10.1002/open.201300018
PMCID: PMC3775520  PMID: 24482763
cancer biomarkers; electrochemistry; inkjet-printed arrays; microfluidics; sensors
7.  A genome-wide RNAi screen reveals a Trio-regulated Rho GTPase circuitry transducing GPCR-initiated mitogenic signals 
Molecular cell  2012;49(1):94-108.
Activating mutations in GNAQ and GNA11, encoding members of the Gαq family of G protein α subunits, are the driver uveal melanoma oncogenes, while mutations in Gq-linked G proteincoupled receptors (GPCRs) have been identified recently in numerous human malignancies. How Gαq and its coupled receptors transduce mitogenic signals is still unclear, due to the complexity of signaling events perturbed upon Gq activation. Using of a synthetic biology approach and a genome-wide RNAi screen, we found that a highly conserved guanine nucleotide exchange factor, Trio, is essential to activate Rho- and Rac-regulated signaling pathways acting on JNK and p38, thereby transducing proliferative signals from Gαq to the nucleus independently of PLC-β. Indeed, while many biological responses elicited by Gq depend on the transient activation of second messenger system, Gq utilizes a hardwired protein-protein interaction-based signaling circuitry to achieve the sustained stimulation of proliferative pathways, thereby controlling normal and aberrant cell growth.
doi:10.1016/j.molcel.2012.10.018
PMCID: PMC3545055  PMID: 23177739
MAPK; JNK; p38; Rho GTPases; Signal Transduction; G Proteins; Cancer
8.  Chemopreventive and chemotherapeutic actions of mTOR inhibitor in genetically-defined head and neck squamous cell carcinoma mouse model 
Purpose
To assess the efficacy of Rapamycin treatment in chemoprevention and chemotherapy of tumorigenesis in a genetically-defined mouse model of head and neck squamous cell carcinoma (HNSCC).
Experimental design
Knockdown of Tgfbr1 and/or Pten using siRNA-mediated RNA interference was carried out in human HNSCC cell lines to analyze molecular changes in the mTOR pathway. Tgfbr1flox/flox; Ptenflox/flox; K14-CreERtam mice were treated with oral gavage of tamoxifen for the conditional deletion of Tgfbr1 and Pten in oral mucosa, resuting in HNSCC (Bian et al 2011). Tgfbr1 and Pten conditonal deletion (2cKO) mice were treated with Rapamycin before or after the onset of HNSCC, and the efficacy of this treatment was assessed by determining tumor burden, longevity, and molecular analysis of the mTOR pathway. Molecular changes observed in human HNSCC cell lines and 2cKO mice were compared to identify key alterations in the mTOR pathway.
Results
Knockdown of Tgfbr1 and/or Pten in human HNSCC cell lines resulted in activation of mTORC1 and increased levels of survivin. Furthermore, we observed similar changes in HNSCC of the 2cKO mouse. In the human HNSCC tissue array, a loss of Tgfbr1 expression correlated with increased survivin levels. Chemopreventive Rapamycin treatment significantly delayed the onset of the HNSCC tumors and prolonged survival in 2cKO mice. Additionally, we also found that Rapamycin had a therapeutic effect on squamous cell carcinomas in these mice. In 2cKO HNSCC tongue tumors, Rapamycin treatment induced apoptosis, inhibited cell proliferation and phosphorylation of Akt and S6, and decreased survivin expression.
Conclusions
These findings indicate that tumorigenesis in 2cKO HNSCC is associated with activation of the Akt/mTOR/survivin pathway, and inhibition of this pathway by Rapamycin treatment successfully ameliorates the onset and progression of tumorigenesis.
doi:10.1158/1078-0432.CCR-12-1371
PMCID: PMC3463723  PMID: 22859719
TGF-β; Pten; head and neck cancer; mTOR; survivin
9.  Preventive and therapeutic effects of Smad7 on radiation-induced oral mucositis 
Nature medicine  2013;19(4):421-428.
We report that K5.Smad7 mice, which express Smad7 transgene by a keratin-5 promoter, were resistant to radiation-induced oral mucositis, a painful oral ulceration. In addition to NF-κB activation known to contribute to oral mucositis, we found activated TGF-β signaling in oral mucositis. Smad7 dampened both pathways to attenuate inflammation, growth inhibition and apoptosis. Additionally, Smad7 promoted oral epithelial migration to close the wound. Further analyses revealed that TGF-β signaling Smads and their co-repressor CtBP1 transcriptionally repressed Rac1, and Smad7 abrogated this repression. Knocking down Rac1 in mouse keratinocytes abrogated Smad7-induced migration. Topically applying Smad7 protein with a cell permeable Tat-tag (Tat-Smad7) to oral mucosa showed preventive and therapeutic effects on radiation-induced oral mucositis in mice. Thus, we have identified novel molecular mechanisms involved in oral mucositis pathogenesis and our data suggest an alternative therapeutic strategy to block multiple pathological processes of oral mucositis.
doi:10.1038/nm.3118
PMCID: PMC3780964  PMID: 23475202
10.  mTOR inhibition prevents epithelial stem cell senescence and protects from radiation-induced mucositis 
Cell stem cell  2012;11(3):401-414.
SUMMARY
The integrity of the epidermis and mucosal epithelia is highly dependent on resident self-renewing stem cells, which makes them vulnerable to physical and chemical insults compromising the repopulating capacity of the epithelial stem cell compartment. This is frequently the case in cancer patients receiving radiation or chemotherapy, many of whom develop mucositis, a debilitating condition involving painful and deep mucosal ulcerations. Here, we show that inhibiting the mammalian target of rapamycin (mTOR) with rapamycin increases the clonogenic capacity of primary human oral keratinocytes and their resident self-renewing cells by preventing stem cell senescence. This protective effect of rapamycin is mediated by the increase expression of mitochondrial superoxide dismutase (MnSOD), and the consequent inhibition of ROS formation and oxidative stress. mTOR inhibition also protects from the loss of proliferative basal epithelial stem cells upon ionizing radiation in vivo, thereby preserving the integrity of the oral mucosa and protecting from radiation-induced mucositis.
doi:10.1016/j.stem.2012.06.007
PMCID: PMC3477550  PMID: 22958932
11.  Future Directions and Treatment Strategies for Head and Neck Squamous Cell Carcinomas 
Head and neck cancer is a devastating disease that afflicts many individuals worldwide. Conventional therapies are successful in only a limited subgroup and often leave the patient with disfigurement and long lasting adverse effects on normal physiological functions. The field is in dire need of new therapies. Oncolytic viral as well as targeted therapies have shown some success in other malignancies and are attractive for the treatment of head and neck cancer. Recently, it has been shown that a subset of head and neck cancers is human papillomavirus (HPV) positive and that this subset of cancers is biologically distinct and more sensitive to chemoradiation therapies although the underlying mechanism is unclear. However, chemoresistance remains a general problem. One candidate mediator of therapeutic response, which is of interest for the targeting of both HPV-positive and -negative tumors is the human DEK proto-oncogene. DEK is upregulated in numerous tumors including head and neck cancers regardless of their HPV status. Depletion of DEK in tumor cells in culture results in sensitivity to genotoxic agents, particularly in rapidly proliferating cells. This suggests that tumors with high DEK protein expression may be correlated with poor clinical response to clastogenic therapies. Targeting molecules such as DEK in combination with new and/or conventional therapies, holds promise for novel future therapeutics for head and neck cancer.
doi:10.1016/j.trsl.2012.02.002
PMCID: PMC3423575  PMID: 22683420
12.  Rapid Microfluidic Immunoassays of Cancer Biomarker Proteins Using Disposable Inkjet-Printed Gold Nanoparticle Arrays 
ChemistryOpen  2013;2(4):141-145.
doi:10.1002/open.201300018
PMCID: PMC3775520  PMID: 24482763
cancer biomarkers; electrochemistry; inkjet-printed arrays; microfluidics; sensors
13.  Ultrasensitive Detection of Cancer Biomarkers in the Clinic using a Nanostructured Microfluidic Array 
Analytical chemistry  2012;84(14):6249-6255.
Multiplexed biomarker protein detection holds unrealized promise for clinical cancer diagnostics due to lack of suitable measurement devices and lack of rigorously validated protein panels. Here we report an ultrasensitive electrochemical microfluidic array optimized to measure a four-protein panel of biomarker proteins, and we validate the protein panel for accurate oral cancer diagnostics. Unprecedented ultralow detection into the 5–50 fg mL−1 range was achieved for simultaneous measurement of proteins IL-6, IL-8, VEGF and VEGF-C in diluted serum. The immunoarray achieves high sensitivity in 50 min assays by using off-line protein capture by magnetic beads carrying 400,000 enzyme labels and 120,000 antibodies. After capturing the proteins and washing to inhibit non-specific binding, the beads are magnetically separated and injected into the array for selective capture by antibodies on eight nanostructured sensors. Good correlations with ELISA for protein determinations in conditioned cancer cell media confirmed the accuracy of this approach. Normalized means of the 4-protein levels in 78 oral cancer patient serum samples and 49 controls gave clinical sensitivity 89% and specificity 98% for oral cancer detection, demonstrating high diagnostic utility. The low cost, easily fabricated immunoarray provides a rapid serum test for diagnosis and personalized therapy of oral cancer. The device is readily adaptable to clinical diagnostics of other cancers.
doi:10.1021/ac301392g
PMCID: PMC3418660  PMID: 22697359
14.  Loss of TGF-β Signaling and PTEN Promotes Head and Neck Squamous Cell Carcinoma through Cellular Senescence Evasion and Cancer-related Inflammation 
Oncogene  2011;31(28):3322-3332.
The molecular mechanisms that contribute to the initiation and progression of head and neck squamous cell carcinoma (HNSCC) have not been completely delineated. Our observations indicate that defects in the TGF-β and PI3K/Akt signaling pathways are common in human HNSCCs. Conditional activation of the PI3K/Akt pathway due to Pten deletion in the mouse head and neck epithelia gives rise to hyperproliferation, but only a few lesions progress to HNSCC. However, Pten-deficient mice developed full-penetrance HNSCC in combination with type I TGF-β receptor (Tgfbr1) deletion. Molecular analysis revealed enhanced cell proliferation, decreased apoptosis, and increased expression of CCND1 in the basal layer of the head and neck epithelia, as well as in the tumors of Tgfbr1/Pten double conditional knockout (2cKO) mice. Furthermore, neoplastic transformation involves senescence evasion and is associated with an increased number of putative cancer stem cells (CSCs). In addition, the NF-κB pathway activation, myeloid derived suppressor cell (MDSC) infiltration, angiogenesis, and immune suppression in the tumor microenvironment, all of which are characteristic of human HNSCCs, contribute significantly to head and neck carcinogenesis in 2cKO mice. These tumors display pathology and multiple molecular alterations resembling human HNSCCs. This suggests that the Tgfbr1/Pten 2cKO mouse model is suitable for preclinical intervention, and that it has significant implications in the development of diagnostic cancer biomarkers and effective strategies for prevention and treatment of HNSCCs.
doi:10.1038/onc.2011.494
PMCID: PMC3270146  PMID: 22037217
TGF-β; PI3K/Akt; Head and Neck Squamous Cell Carcinoma (HNSCC); Conditional Knockout; Cancer Mouse Model
15.  Nuclear Mapping of Nano-Drug Delivery Systems in Dynamic Cellular Environments 
ACS Nano  2012;6(6):4966-4972.
Nanoformulations have shown great promise for delivering chemotherapeutics and hold tremendous clinical relevance. However nuclear mapping of the chemo drugs is important to predict the success of the nanoformulation. Herein in this study fluorescence microscopy and a subcellular tracking algorithm were used to map the diffusion of chemotherapeutic drugs in cancer cells. Positively charged nanoparticles efficiently carried the chemo drug across the cell membrane. The algorithm helped map free drug and drug loaded nanoparticles, revealing varying nuclear diffusion pattern of the chemotherapeutics in drug-sensitive and resistant cells in a live dynamic cellular environment. While the drug-sensitive cells showed an exponential uptake of the drug with time, resistant cells showed random and asymmetric drug distribution. Moreover nanoparticles carrying the drug remained in the perinuclear region while the drug got accumulated in the cell nuclei. The tracking approach has enabled us to predict the therapeutic success of different nanoscale formulations of doxorubicin.
doi:10.1021/nn300516g
PMCID: PMC3383922  PMID: 22540867
iron oxide nanoparticles; doxorubicin; drug resistance; computational; nuclear mapping; live cell imaging; cancer
16.  Improved Clearance during Treatment of HPV-Positive Head and Neck Cancer through mTOR Inhibition12 
Neoplasia (New York, N.Y.)  2013;15(6):620-630.
Human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) incidence is increasing at a near epidemic rate. We investigated whether the mammalian (or mechanistic) target of rapamycin (mTOR) inhibitor, rapamycin, can be used as a concurrent agent to standard-of-care cisplatin/radiation therapy (CRT) to attenuate tumor lactate production, thus enhancing CRT-induced immune-mediated clearance of this antigenic tumor type. A C57Bl/6-derived mouse oropharyngeal epithelial cell line retrovirally transduced with HPV type 16 E6/E7 and human squamous cell carcinoma cell lines were evaluated for their response to rapamycin in vitro with proliferation assays, Western blots, and lactate assays. Clonogenic assays and a preclinical mouse model were used to assess rapamycin as a concurrent agent to CRT. The potential of rapamycin to enhance immune response through lactate attenuation was assessed using quantitative tumor lactate bioluminescence and assessment of cell-mediated immunity using E6/E7-vaccinated mouse splenocytes. Rapamycin alone inhibited mTOR signaling of all cancer cell lines tested in vitro and in vivo. Furthermore, rapamycin administered alone significantly prolonged survival in vivo but did not result in any long-term cures. Given concurrently, CRT/rapamycin significantly enhanced direct cell killing in clonogenic assays and prolonged survival in immunocompromised mice. However, in immunocompetent mice, concurrent CRT/rapamycin increased long-term cures by 21%. Preliminary findings suggest that improved survival involves increased cell killing and enhanced immune-mediated clearance in part due to decreased lactate production. The results may provide rationale for the clinical evaluation of mTOR inhibitors concurrent with standard-of-care CRT for treatment of HPV-positive HNSCC.
PMCID: PMC3664994  PMID: 23730210
17.  mTOR as a Molecular Target in HPV-Associated Oral and Cervical Squamous Carcinomas 
Purpose
The incidence of head and neck squamous cell carcinomas (HNSCC) associated with papillomavirus (HPV) infection has increased over the past decades in the US. We aimed at examining the global impact of HPV-associated HNSCC, and whether the established key role of mTOR activation in HNSCC is also observed in HPV+ HNSCC lesions, thereby providing novel treatment options for HPV-associated HNSCC patients.
Experimental Design
An international HNSCC tissue microarray (TMA) was used to analyze the expression of p16INK4A, a surrogate for HPV infection, and Akt-mTOR pathway activation. Results were confirmed in a large collection of HPV− and HPV+ HNSCC cases and in a cervical cancer (CCSCC) TMA. Observations were validated in HNSCC and CCSCC-derived cell lines, which were xenografted into immunodeficient mice for tumorigenesis assays.
Results
Approximately 20% of all HNSCC lesions could be classified as HPV+, irrespective of their country of origin. mTOR pathway activation was observed in most HPV+ HNSCC and CCSCC lesions and cell lines. The pre-clinical efficacy of mTOR inhibition by rapamycin and RAD001 was explored in HPV+ HNSCC and CCSCC tumor xenografts. Both mTOR inhibitors effectively decreased mTOR activity in vivo, and caused a remarkable decrease in tumor burden. These results emphasize the emerging global impact of HPV-related HNSCCs, and indicate that the activation of the mTOR pathway is a widespread event in both HPV− and HPV-associated HNSCC and CCSCC lesions.
Conclusions
The emerging results may provide a rationale for the clinical evaluation of mTOR inhibitors as a molecular targeted approach for the treatment of HPV-associated malignancies.
doi:10.1158/1078-0432.CCR-11-2824
PMCID: PMC3443560  PMID: 22409888
Oral cancer; cervical cancer Human papilloma virus; rapalogs; targeted therapy; mTOR
18.  Metformin prevents the development of oral squamous cell carcinomas from carcinogen-induced premalignant lesions 
Head and neck squamous cell carcinoma (HNSCC) is a major public health concern. The recent identification of the mammalian Target of Rapamycin Complex 1 (mTORC1) signaling pathway as a highly prevalent molecular signature underlying HNSCC pathogenesis has provided the foundation to search for novel therapeutic approaches to prevent and treat HNSCC. Here, we asked whether metformin, the most widely used medication for the treatment of type 2 diabetes, which acts in part by stimulating the AMP-activated protein kinase (AMPK) signaling pathway thereby reducing mTORC1 activity, may lower the risk of HNSCC development. Indeed, we show that metformin reduces the growth of HNSCC cells and diminishes their mTORC1 activity by both AMPK-dependent and –independent mechanisms. We also optimized an oral-specific carcinogenesis mouse model that results in the accumulation of multiple oral premalignant lesions at the end of the carcinogen exposure, some of which then spontaneously progress into HNSCC. Using this mouse model, we observed that metformin specifically inhibits mTORC1 in the basal proliferating epithelial layer of oral premalignant lesions. Remarkably, metformin prevented the development of HNSCC by reducing significantly the size and number of carcinogen-induced oral tumoral lesions, and by preventing their spontaneous conversion to squamous cell carcinomas. Collectively, our data underscore the potential clinical benefits of using metformin as a targeted chemopreventive agent in the control of HNSCC development and progression.
doi:10.1158/1940-6207.CAPR-11-0502
PMCID: PMC3429367  PMID: 22467081
Oral cancer; premalignant lesions; targeted therapy; mTOR
20.  TRAIL induces apoptosis in oral squamous carcinoma cells: a crosstalk with oncogenic Ras regulated cell surface expression of death receptor 5 
Oncotarget  2013;4(2):206-217.
TNF-related apoptosis inducing ligand (TRAIL) induces apoptosis through its death receptors (DRs) 4 and/or 5 expressed on the surface of target cells. The selectivity of TRAIL towards cancer cells has promoted clinical evaluation of recombinant human TRAIL (rhTRAIL) and its agonistic antibodies in treating several major human cancers including colon and non-Hodgkin's lymphoma. However, little is known about their ability in killing oral squamous cell carcinoma (OSCC) cells. In this study, we tested the apoptotic responses of a panel of seven human OSCC cell lines (HN31, HN30, HN12, HN6, HN4, Cal27, and OSCC3) to rhTRAIL and monoclonal antibodies against DR4 or DR5. We found that rhTRAIL is a potent inducer of apoptosis in most of the oral cancer cell lines tested both in vitro and in vivo. We also showed that DR5 was expressed on the surface of the tested cell lines which correlated with the cellular susceptibility to apoptosis induced by rhTRAIL and anti-DR5 antibody. By contrast, little or no DR4 was detected on the surface of OSCC3 and HN6 cells rendering cellular resistance to DR4 antibody and a reduced sensitivity to rhTRAIL. Notably, the overall TRAIL sensitivity correlated well with the levels of endogenous active Ras in the cell lines tested. Expression of a constitutively active Ras mutant (RasV12) in OSCC3 cells selectively upregulated surface expression of DR5, but not DR4, and restored TRAIL sensitivity. Our findings could have implications for the use of TRAIL receptor targeted therapies in the treatment of human OSCC tumors particularly the ones harboring constitutively active Ras mutant.
PMCID: PMC3712567  PMID: 23470485
oral cancer; TRAIL; death receptors; apoptosis; Ras; oncotarget
21.  Semaphorin signaling in angiogenesis, lymphangiogenesis and cancer 
Cell Research  2011;22(1):23-32.
Angiogenesis, the formation of new blood vessels from preexisting vasculature, is essential for many physiological processes, and aberrant angiogenesis contributes to some of the most prevalent human diseases, including cancer. Angiogenesis is controlled by delicate balance between pro- and anti-angiogenic signals. While pro-angiogenic signaling has been extensively investigated, how developmentally regulated, naturally occurring anti-angiogenic molecules prevent the excessive growth of vascular and lymphatic vessels is still poorly understood. In this review, we summarize the current knowledge on how semaphorins and their receptors, plexins and neuropilins, control normal and pathological angiogenesis, with an emphasis on semaphorin-regulated anti-angiogenic signaling circuitries in vascular and lymphatic endothelial cells. This emerging body of information may afford the opportunity to develop novel anti-angiogenic therapeutic strategies.
doi:10.1038/cr.2011.198
PMCID: PMC3351930  PMID: 22157652
semaphorin; signaling; angiogenesis; lymphangiogenesis; cancer
22.  Signaling Circuitries Controlling Stem Cell Fate: To Be or Not To Be 
Current Opinion in Cell Biology  2011;23(6):716-723.
The integration of extrinsic and intrinsic signals is required to preserve the self-renewal and tissue regenerative capacity of adult stem cells, while protecting them from malignant conversion or loss of proliferative potential by death, differentiation or senescence. Here we review emerging signaling circuitries regulating stem cell fate, with emphasis on epithelial stem cells. Wnt, mTOR, GPCRs, Notch, Rho GTPases, YAP and DNA and histone methylases are some of the mechanisms that allow stem cells to balance their regenerative potential and the initiation of terminal differentiation programs, guaranteeing appropriate tissue homeostasis. Understanding the signaling circuitries regulating stem cell fate decisions might provide important insights into cancer initiation and numerous human pathologies that involve the progressive loss of tissue-specific adult stem cells.
doi:10.1016/j.ceb.2011.08.002
PMCID: PMC3391582  PMID: 21880478
23.  Decreased Lymphangiogenesis and Lymph Node Metastasis by mTOR Inhibition in Head and Neck Cancer 
Cancer research  2011;71(22):7103-7112.
Despite our improved understanding of cancer, the 5-year survival rate for head and neck squamous cell carcinomas (HNSCC) patients remains relatively unchanged at 50% for the past three decades. HNSCC often metastasize to locoregional lymph nodes, and lymph node involvement represents one of the most important prognostic factors of poor clinical outcome. Among the multiple dysregulated molecular mechanism in HNSCC, emerging basic, preclinical, and clinical findings support the importance of the mTOR signaling route in HNSCC progression. Indeed, we observed here that the activation of mTOR is a widespread event in clinical specimens of HNSCC invading locoregional lymph nodes. We developed an orthotopic model of HNSCC consisting in the implantation of HNSCC cells into the tongues of immunocompromised mice. These orthotopic tumors spontaneously metastasize to the cervical lymph nodes, where the presence of HNSCC cells can be revealed by histological and immunohistochemical evaluation. Both primary and metastatic experimental HNSCC lesions exhibited elevated mTOR activity. The ability to monitor and quantitate lymph node invasion in this model system enabled us to explore whether the blockade of mTOR could impact on HNSCC metastasis. We found that inhibition of mTOR with rapamycin and the rapalog RAD001 diminished lymphangiogenesis in the primary tumors and prevented the dissemination of HNSCC cancer cells to the cervical lymph nodes, thereby prolonging animal survival. These findings may provide a rationale for the future clinical evaluation of mTOR inhibitors, including rapamycin and its analogs, as part of a molecular-targeted metastasis preventive strategy for the treatment of HNSCC patients.
doi:10.1158/0008-5472.CAN-10-3192
PMCID: PMC3443559  PMID: 21975930
mTOR; metastasis; oral cancer; targeted therapies; signal transduction
24.  Nuclear factor of activated T-cell c3 inhibition of mammalian target of rapamycin signaling through induction of regulated in development and DNA damage response 1 in human intestinal cells 
Molecular Biology of the Cell  2012;23(15):2963-2972.
Our results demonstrate that nuclear factor of activated T-cell 3 (NFATc3) contributes to the regulation of the mammalian target of rapamycin (mTOR) repressor regulated in development and DNA damage response 1 (REDD1) and mTOR downstream-targeted c-Myc expression. Furthermore, our study demonstrates a novel role for the NFATc3/REDD1/tuberous sclerosis complex 2 axis in the regulation of goblet cell differentiation.
The nuclear factor of activated T-cell (NFAT) proteins are a family of transcription factors (NFATc1–c4) involved in the regulation of cell differentiation. We identified REDD1, a negative regulator of mammalian target of rapamycin (mTOR) through the tuberous sclerosis complex (TSC1/2 complex), as a new molecular target of NFATc3. We show that treatment with a combination of phorbol 12-myristate 13-acetate (PMA) plus ionophore A23187 (Io), which induces NFAT activation, increased REDD1 mRNA and protein expression and inhibited mTOR signaling; pretreatment with the calcineurin inhibitor cyclosporin A (CsA), an antagonist of NFAT signaling, decreased REDD1 induction and mTOR inhibition. Knockdown of NFATc3, not NFATc1, NFATc2, or NFATc4, attenuated PMA/Io-induced REDD1 expression. Treatment with PMA/Io increased REDD1 promoter activity and increased NFATc3 binding to the REDD1 promoter. Overexpression of NFATc3 increased REDD1 mRNA and protein expression and increased PMA/Io-mediated REDD1 promoter activity. Treatment with PMA/Io increased expression of the goblet cell differentiation marker MUC2; these changes were attenuated by pretreatment with CsA or knockdown of REDD1 or NFATc3. Overexpression of NFATc3 increased, while knockdown of TSC2 decreased, MUC2 expression. We provide evidence showing NFATc3 inhibits mTOR via induction of REDD1. Our results suggest a role for the NFATc3/REDD1/TSC2 axis in the regulation of intestinal cell differentiation.
doi:10.1091/mbc.E12-01-0037
PMCID: PMC3408422  PMID: 22696685
25.  The Biology of Tobacco and Nicotine: Bench to Bedside 
Strong epidemiologic evidence links smoking and cancer. An increased understanding of the molecular biology of tobacco-related cancers could advance progress toward improving smoking cessation and patient management. Knowledge gaps between tobacco addiction, tumorigenesis, and cancer brought an interdisciplinary group of investigators together to discuss “The Biology of Nicotine and Tobacco: Bench to Bedside.” Presentations on the signaling pathways and pathogenesis in tobacco-related cancers, mouse models of addiction, imaging and regulation of nicotinic receptors, the genetic basis for tobacco carcinogenesis and development of lung cancer, and molecular mechanisms of carcinogenesis were heard. Importantly, new opportunities to use molecular biology to identify and abrogate tobacco-mediated carcinogenesis and to identify high-risk individuals were recognized.
doi:10.1158/1055-9965.EPI-04-0652
PMCID: PMC3459058  PMID: 15824140

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