Rolando E. Mendez

MASSIVE SATELLITES OF CLOSE-IN GAS GIANT EXOPLANETS

We study the orbits, tidal heating and mass loss from satellites around close-in gas giant exoplanets. The focus is on large satellites which are potentially observable by their transit signature. We argue that even Earth-size satellites around hot Jupiters can be immune to destruction by orbital decay; detection of such a massive satellite would strongly constrain theories of tidal dissipation in gas giants, in a manner complementary to orbital circularization. The stars gravity induces significant periodic eccentricity in the satellites orbit. The resulting tidal heating rates, per unit mass, are far in excess of Ios and dominate radioactive heating out to planet orbital periods of months for reasonable satellite tidal Q. Inside planet orbital periods of about a week, tidal heating can completely melt the satellite. Lastly, we compute an upper limit to the satellite mass loss rate due to thermal evaporation from the surface, valid if the satellites atmosphere is thin and vapor pressure is negligible. Using this upper limit, we find that although rocky satellites around hot Jupiters with orbital periods less than a few days can be significantly evaporated in their lifetimes, detectable satellites suffer negligible mass loss at longer orbital periods.

Activation of the insulin-like growth factor-1 receptor alters p27 regulation by the epidermal growth factor receptor in oral squamous carcinoma cells.

BACKGROUND Although oral squamous cell carcinomas (OSCCs) commonly overexpress the epidermal growth factor receptor (EGFR), EGFR tyrosine kinase inhibitors (TKIs) exhibit poor efficacy clinically. Activation of the insulin-like growth factor-1 receptor (IGF1R) induces resistance of OSCC cells to EGFR-TKIs in vitro. This study seeks to evaluate the changes in cell cycle status in OSCC cells in response to gefitinib and IGF1R activation. METHODS SCC-25 OSCC cells were used for in vitro analyses. RESULTS Gefitinib caused a 50% reduction in S-phase population, and IGF1R activation caused a 2.8-fold increase; combined treatment yielded a baseline S-phase population. Gefitinib treatment increased the cyclin-dependent kinase inhibitor p27, and this was not abrogated by IGF1R activation. pT157-p27 was noted by immunoblot to be decreased on gefitinib treatment, but this was reversed with IGF1R activation. T157 phosphorylation contributes to cytoplasmic localization of p27 where it can promote cell proliferation and cell motility. Using both subcellular fractionation and immunofluorescence microscopy techniques, IGF1R stimulation was noted to increase the relative cytoplasmic localization of p27; this persisted when combined with gefitinib. CONCLUSIONS IGF1R activation partially reverses the cell cycle arrest caused by gefitinib in OSCC cells. While IGF1R stimulation does not eliminate the gefitinib-induced increase in total p27, its phosphorylation state and subcellular localization are altered. This may contribute to the ability of the IGF1R to rescue OSCC cells from EGFR-TKI treatment and may have important implications for the use of p27 as a biomarker of cell cycle arrest and response to therapy.

p70S6 kinase is a critical node that integrates HER-family and PI3 kinase signaling networks.

Therapies targeting oncogenic drivers rapidly induce compensatory adaptive responses that blunt drug effectiveness, contributing to therapeutic resistance. Adaptive responses are characteristic of robust cell signaling networks, and thus there is increasing interest in drug combinations that co-target the driver and the adaptive response. An alternative approach to co-inhibiting oncogenic and adaptive targets is to identify a critical node where the activities of these targets converge. Nodes of convergence between signaling modules represent potential therapeutic vulnerabilities because their inhibition could result in the collapse of the network, leading to enhanced cytotoxicity. In this report we demonstrate that p70S6 kinase (p70S6K) can function as a critical node linking HER-family and phosphoinositide-3-kinase (PI3K) pathway signaling. We used high-throughput combinatorial drug screening to identify adaptive survival responses to targeted therapies, and found that HER-family and PI3K represented compensatory signaling pathways. Co-targeting these pathways with drug combinations caused synergistic cytotoxicity in cases where inhibition of neither target was effective as a monotherapy. We utilized Reverse Phase Protein Arrays and determined that phosphorylation of ribosomal protein S6 was synergistically down-regulated upon HER-family and PI3K/mammalian target of rapamycin (mTOR) co-inhibition. Expression of constitutively active p70S6K protected against apoptosis induced by combined HER-family and PI3K/mTOR inhibition. Direct inhibition of p70S6K with small molecule inhibitors phenocopied HER-family and PI3K/mTOR co-inhibition. These data implicate p70S6K as a critical node in the HER-family/PI3K signaling network. The ability of direct inhibitors of p70S6K to phenocopy co-inhibition of two upstream signaling targets indicates that identification and targeting of critical nodes can overcome adaptive resistance to targeted therapies.

Effect of Adipose-Derived Stem Cells on Head and Neck Squamous Cell Carcinoma

Objective Patients with head and neck squamous cell carcinoma (HNSCC) have significant wound-healing difficulties. While adipose-derived stem cells (ASCs) facilitate wound healing, ASCs may accelerate recurrence when applied to a cancer field. This study evaluates the impact of ASCs on HNSCC cell lines in vitro and in vivo. Study Design In vitro experiments using HNSCC cell lines and in vivo mouse experiments. Setting Basic science laboratory. Subjects and Methods Impact of ASCs on in vitro proliferation, survival, and migration was assessed using 8 HNSCC cell lines. One cell line was used in a mouse orthotopic xenograft model to evaluate in vivo tumor growth in the presence and absence of ASCs. Results Addition of ASCs did not increase the number of HNSCC cells. In clonogenic assays to assess cell survival, addition of ASCs increased colony formation only in SCC9 cells (maximal effect 2.3-fold, P < .02) but not in other HNSCC cell lines. In scratch assays to assess migration, fluorescently tagged ASCs did not migrate appreciably and did not increase the rate of wound closure in HNSCC cell lines. Addition of ASCs to HNSCC xenografts did not increase tumor growth. Conclusion Using multiple in vitro and in vivo approaches, ASCs did not significantly stimulate HNSCC cell proliferation or migration and increased survival in only a single cell line. These findings preliminarily suggest that the use of ASCs may be safe in the setting of HNSCC but that further investigation on the therapeutic use of ASCs in the setting of HNSCC is needed.

Abstract 5633: Co-targeting the IGF1R pathway and compensatory signaling enhances cytotoxicity in head and neck cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The 5-year survival rate for patients with cancer of the Head and Neck (HNC) has not significantly improved over the past decade. Clearly, new therapeutic targets and strategies for employing existing targeted and cytotoxic therapies are needed. Studies have shown both expression and phosphorylation of the insulin-like growth factor 1 receptor (IGF1R) in both head and neck cancer cell lines and patient tumor samples. We found that small molecule inhibitors targeting IGF1R, when used as a single agent, caused varying degrees of cytotoxicity in HNC cell lines. In order to determine whether the variable biological response to IGF1R inhibition was caused by differences in the effects of the IGF1R inhibitors on the cell signaling networks of the HNC cell lines, we performed reverse phase proteomic array analysis on a panel of HNC cell lines treated with IGF1R inhibitors. Preliminary results suggest that treatment with IGF1R inhibitors caused increased expression and/or phosphorylation of a number of proteins in the array. We hypothesize that these alterations represent compensatory signaling pathways that provide resistance to cytotoxicity upon treatment with IGF1R inhibitors. In order to identify possible mechanisms of compensation for the loss of IGF1R pathway signaling, we screened a panel of small molecule inhibitors of the IGF1R/PI3K/AKT signaling pathway against a panel of inhibitors of proteins important in HNC cell signaling Preliminary results indicate that the inhibition of epigenetic modifying proteins as well as members of canonical cell signaling pathways in combination with inhibition of IGF1R signaling leads to a synergistic increase in cytotoxicity. This suggests that compensatory mechanisms exist in HNC that serve to blunt the cytotoxic effect of inhibition of IGF1R. Co-targeting IGF1R and members of these compensatory pathways may be a viable therapeutic strategy. Citation Format: Mark J. Axelrod, Daniel Gioeli, Elizabeth R. Sharlow, Mark C. Conaway, Rolando E. Mendez, Ashraf Khalil, Linnea Taniguchi, Emmanual F. Petricoin, Stephanie Leimgruber, Michael J. Weber, Mark J. Jameson. Co-targeting the IGF1R pathway and compensatory signaling enhances cytotoxicity in head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5633. doi:10.1158/1538-7445.AM2013-5633

Synergistic apoptosis in head and neck squamous cell carcinoma cells by co-inhibition of insulin-like growth factor-1 receptor signaling and compensatory signaling pathways

In head and neck squamous cell carcinoma (HNSCC), resistance to single‐agent targeted therapy may be overcome by co‐targeting of compensatory signaling pathways.

Nanoimmunoassay to Detect Responses in Head and Neck Cancer Feasibility in a Mouse Model

Objective To demonstrate the feasibility of detecting and quantifying extracellular signal-related kinase (ERK) phosphorylation status using nanoimmunoassay (NIA). Study Design Analyses using Cal27, SCC25, and OSC19 head and neck squamous carcinoma cell lines in vitro and in a murine xenograft model. Subjects and Methods NIA and immunoblot were performed on whole-cell lysates, tumor lysates, and fine-needle aspirate biopsies to detect ERK phosphorylation states. Results Using NIA, all 6 isoforms of ERK1/2, including nonphosphorylated, monophosphorylated, and diphosphorylated species, could be reliably detected, distinguished, and quantified in a single assay using a single antibody. In vitro treatment of Cal27 cells with the epidermal growth factor receptor inhibitor gefitinib abolished phospho-ERK detection by immunoblot but resulted in residual detectable species by NIA. Residual phospho-ERK in gefitinib-treated cells could be further reduced by the addition of the insulin-like growth factor 1 receptor inhibitor OSI-906; this correlated with an additional decrease in proliferation over gefitinib alone. In a pilot study of 4 murine xenograft tumors, NIA performed on tumor lysates and fine-needle aspirate biopsies demonstrated altered ERK profiles after 2 days of gefitinib treatment compared with untreated mice. Conclusion NIA offers a novel approach to quantitating the activation state of signaling molecules such as ERK in nanoscale in vitro and in vivo samples across a wide dynamic range. As such, it has potential to provide molecular diagnostic information before, during, and after treatment using a minimally invasive technique. Further study is warranted to determine its utility in assessing signaling proteins as biomolecular outcome predictors in clinical trials.

Abstract 902: XRCC1 induction after cisplatin treatment in head and neck squamous carcinoma cell lines: Evaluation using nanoimmunoassay

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The objective of this study was to use nanoimmunoassay (NIA) to quantify XRCC1 levels and correlate these with response to platinum-based chemotherapeutic agents in HNSCC cell lines. NIA is a developing technique that uses nanogram-scale samples to quantify specific proteins by immunoassay after separation by isoelectric focusing. Detection is more sensitive and quantification more accurate than with standard immunoblot techniques. XRCC1 is a base-excision repair protein that has been identified as a possible mediator of resistance to platinum-based chemotherapeutic agents, but its role and predictive utility have undergone limited investigation in head and neck squamous cell carcinoma (HNSCC). Detection of XRCC1 by NIA was first optimized. In lysates of HNSCC cell lines, XRCC1 was consistently identified at a pI of 5.45 ± 0.1. Peak intensity was improved by the addition of 7 M urea to the specimen prior to loading. In order to correct for sample and loading variability, and detection of a simultaneous internal loading control was also optimized. Under conditions that were optimal for detection of XRCC1, thioredoxin as consistently identified at a pI of 4.90 ± 0.1. Using 9 HNSCC cell lines, XRCC1 levels were evaluated using whole cell lysates in the untreated (basal) state as well as 24 h after treatment with cisplatin or vehicle. NIA was performed on 200 ng of whole cell lysate and XRCC1 and thioredoxin peak areas were quantified using the associated software. Proliferation assays were performed using alamarBlue to determine the IC50 for cisplatin in each cell line. Basal XRCC1 expression levels did not correlate with sensitivity to cisplatin. However, XRCC1 levels were noted to be significantly altered after treatment. When XRCC1 levels were normalized to the thioredoxin loading control, the ratio of treated to basal XRCC1 was correlated to the IC50 for cisplatin across all cell lines (R2=0.428). In cell lines identified as resistant to cisplatin, XRCC1 levels increased upon treatment, whereas in sensitive cell lines, levels remained unchanged. These NIA findings were confirmed using XRCC1 immunoblot with beta-actin as a loading control; XRCC1 levels obtained using this approach also correlated with the IC50 for cisplatin (R2=0.518). While timing and methodology need to be further elucidated, the change in XRCC1 protein level in response to a cisplatin challenge has the potential to be used as a biomolecular predictor of sensitivity that could direct treatment modality selection early in the course of therapy for HNSCC patients. The quantities of protein used allow for potential evaluation of fine-needle aspirates in patients undergoing treatment. Citation Format: Stephen S. Schoeff, Dane M. Barrett, James Teng, Ashraf Khalil, Matthew A. Hubbard, Anne K. Maxwell, Amir Allak, Rolando E. Mendez, Mark Axelrod, Mark J. Jameson. XRCC1 induction after cisplatin treatment in head and neck squamous carcinoma cell lines: Evaluation using nanoimmunoassay. [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 902. doi:10.1158/1538-7445.AM2014-902

Abstract 695: p70S6 kinase is a critical node that integrates HER-family and PI3 kinase signaling

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Targeted cancer therapies often induce compensatory adaptive responses that blunt drug effectiveness, resulting in therapeutic resistance - intrinsic or acquired. Adaptive responses are characteristic of the complex nature of the cancer cell signaling network. Within this network, nodes of convergence between signaling modules are critical mediators of adaptive responses to targeted drugs. The critical nodes in an oncogenically-activated signaling network represent potential therapeutic vulnerabilities because their inhibition could result in collapse of the network and hence enhanced cytotoxicity. We have previously used high-throughput combinatorial drug screening to empirically identify adaptive survival responses to targeted therapies. We found that HER-family and PI3K represented compensatory signaling pathways, and combination therapy caused synergistic cytotoxicity in cases where inhibition of neither target was effective as a monotherapy. RPPA analysis identified ribosomal protein S6 as being synergistically down-regulated upon HER-family and PI3K co-inhibition. Expression of a constitutively active construct of the upstream activator of S6, p70S6K, was protective against apoptosis induced by combined HER-family and PI3K inhibition. Direct inhibition of p70S6K using small molecule inhibitors phenocopied the growth inhibition and apoptosis caused by HER-family and PI3K inhibition. These data implicate p70S6K as a critical node and druggable target in the HER-family / PI3K signaling network. The ability of direct inhibitors of p70S6K to phenocopy the co-inhibition of upstream signaling indicates that identification and targeting of critical nodes may be a way to overcome adaptive resistance to targeted therapies. Citation Format: Mark J. Axelrod, Rolando E. Mendez, Daniel G. Gioeli, Mark J. Jameson, Michael J. Weber. p70S6 kinase is a critical node that integrates HER-family and PI3 kinase signaling. [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 695. doi:10.1158/1538-7445.AM2014-695

Abstract 2945: Anti-tumor effect of insulin-like growth factor-1 receptor inhibition in head and neck squamous cell carcinoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Back ground: Insulin-like growth factor-1 receptor (IGF1R) activity is upregulated in a variety of human cancers and IGF1R signaling has been implicated as a mechanism of resistance to various cancer therapies, including radiotherapy, cytotoxic chemotherapy, and targeted molecular therapy. We have demonstrated that IGF1R activation can induce resistance to targeted therapy of head and neck squamous cell carcinomas (HNSCCs) using epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). Targeting the IGF1R by selective small molecule kinase inhibition may thus hold promise as an adjuvant treatment for HNSCC. Aims: (1) To assess the anti-tumor effects of two IGF1R/insulin receptor TKIs, BMS-754807 and OSI-906, in multiple HNSCC cell lines in vitro. (2) To evaluate the effect of combined IGF1R and EGFR inhibition in HNSCC cell lines in vitro. Methods: The effect of IGF1R inhibition on cell proliferation, viability, survival, and apoptosis was assessed using alamarBlue, trypan blue exclusion, clonogenic, and flow cytometric assays in 9 HNSCC cell lines. Alterations in downstream IGF1R signaling were assessed by standard immunoblot. The impact of combining an EGFR-TKI with an IGF1R-TKI was measured. Result: Both BMS-754807 and OSI-906 inhibited IGF1R phosphorylation at 200 nM. Complete inhibition of the phosphorylation of IRS-1 and Akt required higher concentrations. Both inhibitors slowed proliferation, decreased cell viability, and decreased cell survival in all 9 cell lines. The EC50 to inhibit proliferation was in the range of X to X microM for BMS-754807 and X to X microM for OSI-906. Both drugs induced apoptosis in HNSCC tumor cell lines based on flow cytometric analysis of caspase-3 cleavage and PARP cleavage. In combination with gefitinib and lapatinib, BMS-754807 and OSI-906 were capable of synergistic growth inhibition. Conclusion: Inhibition of IGF1R activity using the selective TKIs BMS-754807 and OSI906 has an antitumor effect on HNSCC cells in vitro. These drugs are synergistic with EGFR-TKIs and co-targeting the two receptors may represent an approach to overcoming clinical resistance to targeted anti-growth factor therapy in HNSCC. Citation Format: Ashraf A. Khalil, Mark J. Axelrod, Matthew A. Hubbard, Anne K. Maxwell, Linnea E. Taniguchi, Rolando E. Mendez, Mark J. Jameson. Anti-tumor effect of insulin-like growth factor-1 receptor inhibition in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2945. doi:10.1158/1538-7445.AM2013-2945