Chi-Iou Lin

Autophagy Induction with RAD001 Enhances Chemosensitivity and Radiosensitivity through Met Inhibition in Papillary Thyroid Cancer

Although autophagy is generally considered a prosurvival mechanism that preserves viability, there is evidence that it could drive an alternative programmed cell death pathway in cells with defects in apoptosis. Because the inhibition of autophagic activity promotes resistance to both chemotherapy and external beam radiation in papillary thyroid cancer (PTC), we determined if RAD001, a potent activator of autophagy, improves the efficacy of either therapy. We found that RAD001 increased the expression level of light chain 3-II, a marker for autophagy, as well as autophagosome formation in cell lines and in human PTC ex vivo. RAD001 sensitized PTC to doxorubicin and external beam radiation in a synergistic fashion, suggesting that combination therapy could improve therapeutic response at less toxic concentrations. The effects of RAD001 were abrogated by RNAi knockdown of the autophagy-related gene 5, suggesting that RAD001 acts, in part, by enhancing autophagy. Because the synergistic activity of RAD001 with doxorubicin and external radiation suggests distinct and complementary mechanisms of action, we characterized how autophagy modulates signaling pathways in PTC. To do so, we performed kinome profiling and discovered that autophagic activation resulted in Src phosphorylation and Met dephosphorylation. Src inhibition did not reverse the effects of RAD001, whereas Met inhibition reversed the effects of autophagy blockade on chemosensitivity. These results suggest that the anticancer effects of autophagic activation are mediated largely through Met. We conclude that RAD001 induces autophagy, which enhances the therapeutic response to cytotoxic chemotherapy and external beam radiation in PTC. Mol Cancer Res; 8(9); 1217–26. ©2010 AACR.

Galectin-3 Targeted Therapy with a Small Molecule Inhibitor Activates Apoptosis and Enhances Both Chemosensitivity and Radiosensitivity in Papillary Thyroid Cancer

Although most patients with papillary thyroid cancer (PTC) have favorable outcomes, some have advanced PTC that is refractory to external beam radiation and systemic chemotherapy. Galectin-3 (Gal-3) is a β-galactoside–binding protein with antiapoptotic activity that is consistently overexpressed in PTC. The purpose of this study is to determine if Gal-3 inhibition promotes apoptosis, chemosensitivity, and radiosensitivity in PTC. PTC cell lines (8505-C and TPC-1) and human ex vivo PTC were treated with a highly specific small molecule inhibitor of Gal-3 (Td131_1). Apoptotic activity was determined by flow cytometric analysis as well as caspase-3 and PARP cleavage. The minimum inhibitory concentrations of Td131_1 and doxorubicin were determined, and their combined effects were measured to test for synergistic activity. The effects of Td131_1 on radiosensitivity were determined by a clonogenic assay. Td131_1 promoted apoptosis, improved radiosensitivity, and synergistically enhanced chemosensitivity to doxorubicin in PTC cell lines. In PTC ex vivo, Td131_1 treatment alone induced the cleavage of caspase-3 and PARP. Td131_1 and doxorubicin together activated apoptosis in PTC ex vivo to a greater degree than their combined individual effects. Td131_1 activated apoptosis and had synergistic activity with doxorubicin in PTC. We conclude that Gal-3 targeted therapy is a promising therapeutic strategy for advanced PTC that is refractory to surgery and radioactive iodine therapy. (Mol Cancer Res 2009;7(10):1655–62)

Galectin-3 regulates apoptosis and doxorubicin chemoresistance in papillary thyroid cancer cells

A subset of patients with papillary thyroid cancer (PTC) present with aggressive disease that is refractory to conventional treatment. Novel therapies are needed to treat this group of patients. Galectin-3 (Gal-3) is a beta-galactoside-binding protein with anti-apoptotic activity. Over 30 studies in the last 3 years have reported that Gal-3 is highly expressed in PTC relative to normal thyrocytes. In this study, we show that Gal-3 silencing with RNA interference stimulates apoptosis, while Gal-3 overexpression protects against both TRAIL- and doxorubicin-induced apoptosis in PTC cells. The anti-apoptotic activity and chemoresistance related to Gal-3 function can be partially reversed through the inhibition of the PI3K-Akt pathway, suggesting that Gal-3 acts, at least in part, on the PI3K-Akt axis. These observations support further evaluation of Gal-3 as a potential therapeutic target in patients with aggressive PTC.

Necroptosis is a novel mechanism of radiation-induced cell death in anaplastic thyroid and adrenocortical cancers

BACKGROUND Necroptosis is a recently described mechanism of programmed cellular death. We hypothesize that necroptosis plays an important role in radiation-induced cell death in endocrine cancers. METHODS Thyroid and adrenocortical carcinoma cell lines were exposed to increasing doses of radiation in the presence of necroptosis inhibitor Nec-1 and/or apoptosis-inhibitor zVAD. H295R cells deficient in receptor interacting protein 1 (RIP1), an essential kinase for necroptosis, were used as controls. Survival curves were generated at increasing doses of radiation. RESULTS Nec-1 and zVAD increased cellular survival with increasing doses of radiotherapy in 8505c, TPC-1, and SW13. Both inhibitors used together had an additive effect. At 6 Gy, 8505c, TPC-1, and SW13 cell survival was significantly increased compared to controls by 40%, 33%, and 31% with Nec-1 treatment, by 53%, 47%, and 44% with zVAD treatment, and by 80%, 70%, and 65% with both compounds, respectively (P < .05). H295R showed no change in survival with Nec-1 treatment. The radiobiologic parameter quasithreshold dose was significantly increased in 8505c, TPC-1, and SW13 cells when both Nec-1 and zVAD were used in combination to inhibit necroptosis and apoptosis together, revealing resistance to standard doses of fractionated therapeutic radiation. CONCLUSION Necroptosis contributes to radiation-induced cell death. Future studies should investigate ways to promote the activation of necroptosis to improve radiosensitivity.

Autophagy: a new target for advanced papillary thyroid cancer therapy.

BACKGROUND Autophagy is a conserved response to stress that facilitates cell survival in some contexts and promotes cell death in others. We sought to characterize autophagy in papillary thyroid cancer (PTC), and to determine the effects of autophagy inhibition on chemosensitivity and radiosensitivity. METHODS The human thyroid papillary carcinoma cell lines TPC-1 and 8505-C were treated with doxorubicin or radiation in the presence or absence of the autophagy-specific inhibitor 3-methyladenine (3-MA). RESULTS Although light chain 3 (LC3)-II protein levels were undetectable in normal thyroid and PTC specimens at baseline, doxorubicin exposure induced LC3-II expression and the formation of autophagosomes. Both PTC cell lines expressed low levels of LC3-II under standard conditions. Treatment of these cells with doxorubicin strongly induced LC3-II expression and the formation of autophagosomes; however, doxorubicin-mediated induction of LC3-II was abrogated by 3-MA. Moreover, 3-MA significantly increased the doxorubicin IC(50) in both PTC cell lines. Radiation exposure also induced LC3-II expression. Treatment with 3-MA abrogated the radiation-induced increase in LC3-II in both cell lines and reduced radiosensitivity by 49% and 31% in 8505-C and TPC-1 cells, respectively. CONCLUSION Autophagy inhibition promotes PTC resistance to doxorubicin and radiation. Therefore, autophagy activation may be a useful adjunct treatment for patients with PTC that is refractory to conventional therapy.

Autophagic activation potentiates the antiproliferative effects of tyrosine kinase inhibitors in medullary thyroid cancer

BACKGROUND We hypothesized that autophagy inhibition would enhance the anticancer efficacy of ret protooncogene-targeted therapy in medullary thyroid cancer. METHODS Medullary thyroid cancer-1.1 and TT cells were treated with sunitinib or sorafenib in the presence or absence of everolimus, trehalose, or small interfering RNA directed against autophagy protein 5. RESULTS Sunitinib and sorafenib each robustly induced light chain 3-II expression, indicating autophagy activation. Autophagy protein 5 silencing diminished the antiproliferative effects of sunitinib and sorafenib by 44% (P < .05) and 41% (P < .05), respectively, in medullary thyroid cancer-1.1 cells and by 43% (P < .01) and 39% (P < .05), respectively, in TT cells. In contrast, everolimus increased the antiproliferative effects of sunitinib and sorafenib by 24% (P < .01) and 27% (P < .01), respectively, in medullary thyroid cancer-1.1 cells and by 20% (P < .05) and 23% (P < .05), respectively, in TT cells. Trehalose increased the antiproliferative effects of sunitinib and sorafenib by 26% (P < .01) and 27% (P < .01), respectively, in medullary thyroid cancer-1.1 cells and by 28% (P < .05) and 29% (P < .05), respectively, in TT cells. Autophagy protein 5 silencing abrogated both everolimus- and trehalose-induced increases in tyrosine kinase inhibitor efficacy. CONCLUSION Loss (gain) of autophagy diminishes (improves) the efficacy of sunitinib and sorafenib. Our findings suggest that autophagic activation should be combined with targeted ret protooncogene therapy for patients with advanced medullary thyroid cancer.

Sulindac Reverses Aberrant Expression and Localization of β-Catenin in Papillary Thyroid Cancer Cells with the BRAFV600E Mutation

BACKGROUND Activation of the Wnt/beta-catenin signaling pathway is implicated in thyroid tumorigenesis, and up to 90% of papillary thyroid cancer (PTC) demonstrate aberrant expression of beta-catenin. Nonsteroidal antiinflammatory drugs reverse aberrant beta-catenin expression and localization in colon cancer. In this study, we tested the hypothesis that the nonsteroidal antiinflammatory drug sulindac would reverse aberrant beta-catenin activity in thyroid cancer cells. METHODS beta-catenin protein levels were determined in thyroidectomy specimens from six consecutive patients and in three different thyroid cancer cells lines (8505-C, KTC-1, and TPC-1) by immunoblotting. Cells of 8505-C and KTC-1 harbor the BRAF(V600E) mutation, and TPC-1 has the RET/PTC rearrangement. All cell lines were treated with sulindac (100 microM for up to 72 hours). Protein levels of c-myc and cyclin D1 were detected by immunoblotting, and beta-catenin localization was determined by immunocytochemistry in the PTC cell lines. PCCL3 rat thyroid cells that conditionally overexpress either BRAF(V600E) or RET/PTC were also treated with sulindac. RESULTS All PTC specimens and cell lines expressed high levels of beta-catenin protein and displayed aberrant nuclear and cytoplasmic localization of beta-catenin. Exposure to sulindac for 48 hours reduced beta-catenin expression in 8505-C and KTC-1 cells, but not in TPC-1 cells. Further, sulindac treatment reduced c-myc and cyclin D1 levels in 8505-C and KTC-1 cells, but had no effect in TPC-1 cells. Immunocytochemistry demonstrated that sulindac treatment redistributed beta-catenin from the nucleus to the membrane in 8505-C and KTC-1 cells. However, sulindac did not affect beta-catenin localization in TPC-1 cells. Finally, sulindac was effective in decreasing beta-catenin expression and cellular proliferation in BRAF(V600E)-overexpressing cells, but not in RET/PTC3-overexpressing cells. CONCLUSIONS Taken together, our findings demonstrate that sulindac treatment reverses beta-catenin activity in 8505-C and KTC-1 cell lines with the BRAF(V600E), but not in TPC-1 cells with the RET/PTC mutation. Future studies should investigate the potential for beta-catenin-directed therapy for patients with advanced thyroid cancers.

Strategic combination therapy overcomes tyrosine kinase coactivation in adrenocortical carcinoma

BACKGROUND Coactivation of tyrosine kinase limits the efficacy of tyrosine kinase inhibitors. We hypothesized that a strategic combination therapy could overcome tyrosine kinase coactivation and compensatory oncogenic signaling in patients with adrenocortical carcinoma (ACC). METHODS We profiled 88 tyrosine kinases before and after treatment with sunitinib in H295R and SW13 ACC cells. The effects of monotherapy and strategic combination regimens were determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (ie, MTS) assay. RESULTS The minimum inhibitory concentrations (IC(min)) of sunitinib quenched its primary targets: FLT-3, VEGFR-2, and RET. In contrast, ERK, HCK, Chk2, YES, CREB, MEK, MSK, p38, FGR, and AXL were hyperactivated. Monotherapy with sunitinib or PD98059 at their IC(min) reduced proliferation by 23% and 19%, respectively, in H295R cells and by 25% and 24%, respectively, in SW13 cells. Sunitinib and PD98059 in combination decreased proliferation by 68% and 64% in H295R and in SW13 cells, respectively (P < .05 versus monotherapy). The effects of combination treatment exceeded the sum of the effects observed with each individual agent alone. CONCLUSION We describe the first preclinical model to develop strategic combination therapy to overcome tyrosine kinase coactivation in ACC. Because many tyrosine kinase inhibitors are readily available, this model can be immediately tested in clinical trials for patients with advanced ACC.

Thyroid-specific knockout of the tumor suppressor mitogen-inducible gene 6 activates epidermal growth factor receptor signaling pathways and suppresses nuclear factor-κB activity

BACKGROUND Mitogen-inducible gene 6 (Mig-6) is a putative tumor suppressor gene and prognostic biomarker in papillary thyroid cancer. We hypothesized that Mig-6 knockout would activate pro-oncogenic signaling in mouse thyrocytes. METHODS We performed a thyroid-specific knockout using the Cre/loxP recombinase system. RESULTS Four knockout and 4 control mouse thyroids were harvested at 2 months of age. Immunoblotting confirmed Mig-6 ablation in knockout mice thyrocytes. Epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK) phosphorylation levels were increased in Mig-6 knockout compared to wild-type mice. Total EGFR levels were similar in knockout and wild-type mice. However, EGFR was absent in the caveolae-containing membrane fraction of knockout mice, indicating that Mig-6 depletion is associated with a change in the membrane distribution of EGFR. Although p65 localized to the nucleus in wild-type mice, it was distributed in both cytoplasm and nucleus in knockouts, suggesting that Mig-6 loss decreases p65 activity. CONCLUSION Our results confirm the feasibility of targeted, thyroid-specific gene knockout as a strategy for studying the relevance of specific genes in thyroid oncogenesis. We suggest that the loss of Mig-6 alters the membrane distribution of EGFR, which may limit receptor degradation and activate this oncogenic signaling pathway.

Mitogen-Inducible Gene-6 Is a Multifunctional Adaptor Protein with Tumor Suppressor-Like Activity in Papillary Thyroid Cancer

This article appears in The Journal of Clinical Endocrinology & Metabolism, published December 29, 2010, 10.1210/jc.2010-1800